The acetylcholinesterase activity in the colliculus mainly occurs in two layers and is arranged as a lattice in the intermediate grey layer and as a continuous sheet in the superficial grey layer. Undercutting lesions abolish the lattice in the intermediate grey layer but leave the superficial sheet of activity intact. By contrast the injection of kainic acid into the colliculus leaves the intermediate layer lattice intact while causing a local reduction in the superficial layer. Injections of the retrograde tracer Fluoro-Gold into the colliculus labels cells in the pedunculopontine and laterodorsal tegmental nuclei that contains acetylcholinesterase. Cells in the parabigeminal nucleus are also labelled but these cells contain low levels of cholinesterase. Thus, it is concluded that the lattice in the intermediate layers is mainly dependent on afferents from the laterodorsal tegmental and pedunculopontine nuclei while the sheet in the superficial layers is mainly dependent on intrinsic cells.