The Colne rises perennially from a subterranean river at a spring in North Mymms Park in Hertfordshire. Two occasionally dry tributaries: an unnamed brook and the Mimmshall Brook start 2.5 to 4 mi (4.0 to 6.4 km) from the source at golf courses in Barnet, at Borehamwood and in fields by Northaw Place, Northaw. This underground stretch under the old village centre of North Mymms runs for 1.1 mi (1.8 km)
 from swallow holes of the two brooks in the part of that parish named Water End. From the Water End Swallow Holes the Colne runs NW then SW, bounding central and residential Watford and Oxhey, also bounding Rickmansworth and Batchworth, then marking the border between Greater London and Buckinghamshire. After these conjoined settlements it turns south, its main direction, and passes between West Hyde, near Maple Cross, and Harefield, passes Denham Green then passes Uxbridge, where it parallels the Grand Union Canal and its distributary the Frays River which is joined later by the River Pinn between Cowley and Yiewsley on the Greater London side. The Colne Brook splits off as a distributary between New Denham and Uxbridge. After this, at West Drayton, the Frays River rejoins and the Wraysbury River and Duke of Northumberland's River divide off.
In its lower reaches, the river at Longford supplies water to the Longford River, a 12-mile (19 km) artificial channel created in 1610 for King Charles I to supply the water features in Bushy Park and the rectangular lakes in Hampton Court Park.
The Colne's drainage basin extends almost as far north again as the main stream of the river beginning in Hertfordshire: its tributaries, including the Gade, Ver and Misbourne, all extend well into the Chilterns. The Colne's drainage basin is bordered by a range of Thames tributaries to the east and west, including the Crane, Brent and Lea to the east and Wye to the west. To the north of Tring a modest ridge acts as a watershed, separating the Colne's drainage basin from that of the River Great Ouse, which flows north and east towards the Wash.
Eponymous settlements and indirect reference to the river within a district name
The villages of Colney Heath east of St Albans, London Colney south of St Albans, Colney Street (almost wholly a large business park) north of Radlett and Colnbrook between Slough and Heathrow take their name from the river. Three Rivers district covers an area of Hertfordshire named after the River Colne and its two tributary rivers which join the closest together, the River Chess and the River Gade.
Among early proposals to link the river to London was one in 1641 by Sir Edward Forde for a navigable canal, the main purpose of which seems to have been the supply of clean water, and two in 1766, for canals from Marylebone to the river at Uxbridge and another from Marylebone to West Drayton.
The river underwent considerable change in the 1790s, when the Grand Junction Canal (which became part of the Grand Union Canal in 1929) was routed along the valley and still takes the river channel for part of its course. Construction began from Brentford, where it used for 3 miles (4.8 km) the channel of the River Brent, and progressed westwards then northwards, with it reaching Uxbridge in November 1794. To reach the Midlands it continued along the river channel until its junction with the River Gade, which it ran parallel to, but did not take over the channel. The Slough Arm was built in 1882 which required three aqueducts to carry it close to its junction with the Grand Junction, to cross the Fray's River, the River Colne and the Colne Brook.
Between Croxley Green, where the River Gade joins the Colne, and Thorney/West Drayton, below the Slough Arm, the river channels thread their way between many large lakes, some of which were once watercress beds, some chalk pits, and some of which were the result of brickmaking, an industry that developed over several miles of the valley after 1800, when the Grand Junction Canal company advertised the presence of good brick earth, discovered during the construction of the canal.