Immunohistochemistry for choline acetyltransferase (ChAT) revealed an extensive network of cholinergic fibers in the tectum of amphibians. The distribution of ChAT immunoreactive fibers was not restricted to superficial retinocipient layers, but also included deep tectal layers. The aim of the present study was to investigate the origin of the cholinergic inputs to the tectum of amphibians. For that purpose, application of retrograde tracers in the tectum of the anuran Rana perezi and the urodele Pleurodeles waltl was combined with ChAT immunohistochemistry. Double-labeled cells were found primarily in the nucleus isthmi of both species. The cholinergic isthmotectal projection is bilateral and topographically arranged and all retrogradely labeled cells found in this nucleus were ChAT immunoreactive. Remarkably, abundant cholinergic cells in two tegmental nuclei, the pedunculopontine tegmental nucleus (anurans) and the laterodorsal tegmental nucleus (anurans and urodeles), were demonstrated to provide additional cholinergic innervation to the tectum. We compare the present results with previously reported studies in amphibians and other vertebrates, and discuss the possible functional significance of the cholinergic innervation of the amphibian tectum.