Immunocytochemical mapping of cholinergic neurones in the CNS of the cockroach Periplaneta americana has been attempted using monoclonal antibodies to choline acetyltransferase (ChAT, acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6). Monoclonal antibodies 11 255 and 1E6 raised against rat brain ChAT and 1C8 raised against Drosophila melanogaster ChAT were ineffective in staining Periplaneta neurones. However, the cytoplasm of certain neuronal cell bodies was stained by monoclonal antibody 4D7 prepared against rat ChAT. Staining of cell bodies by 4D7 was enhanced following in vivo pre-treatment with colchicine. The staining of specific neurones by monoclonal antibody 4D7 indicates that these cockroach cells are rich in a protein with antigenic determinants resembling those of vertebrate ChAT. For some unidentified neurones, 4D7 staining is associated with the presence of acetylcholinesterase indicating that this monoclonal antibody offers a probe for mapping cholinergic neurones in the CNS of Periplaneta americana. The fast coxal depressor motoneurone (D(f)) was not stained by monoclonal antibody 4D7. Some neuronal processes in the sixth abdominal ganglion, and sensory cell bodies in the cerci were lightly stained by monoclonal antibody 4D7 following pre-injection of animals for 36 hr with colchicine.