The coexistence of immunoreactivities for choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in some brain regions of the rat, using the peroxidase-antiperoxidase method. Consecutive 40 micron thick vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for ChAT and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the retina, cerebral cortex, basal forebrain and spinal cord, colocalization of ChAT-like and GAD-like or GABA-like immunoreactivities was observed in some cell types, whereas no such colocalization was observed in cells in the striatum or brainstem. In the retina, the majority of ChAT-like immunoreactive (ChAT-LI) amacrine cells contained GABA-like or GAD-like immunoreactivity. About half of the ChAT-LI neurons in the cerebral cortex showed GABA-like immunoreactivity. In the basal forebrain only a small proportion of ChAT-LI neurons (0.6%) contained GAD-like immunoreactivity. In the spinal cord, about one-third of ChAT-LI central canal cluster cells and about half of ChAT-LI dorsal horn cells showed GAD-like and/or GABA-like immunoreactivities. These observations indicate the possible coexistence of two classical transmitters, GABA and acetylcholine, in various brain regions and spinal cord of the rat.