Acetylcholine (ACh) conjugates were injected into AKR and DBA mice over a period of 10 weeks. The polyclonal antisera were tested at various immunization times for affinity and specificity using an enzyme-linked immunosorbent assay (ELISA). The most immunoreactive compound was found to be choline-glutaryl-bovine serum albumin (or conjugated ACh). The AKR and DBA mice yielding the highest apparent affinity were killed, and the spleen cells were fused with X63 or SP2/O/Ag mouse myeloma cells. Supernatants of confluent cultures were tested for the presence of anti-conjugated ACh antibodies using the same ELISA method. The best results were obtained with the hybridomas from AKR spleen cells and X63 mouse myeloma cells. Monoclonal antibody affinity and specificity were then evaluated by a radioimmunological procedure using iodinated monoclonal anti-conjugated ACh antibody. From competition experiments, the most immunoreactive compound was choline-glutaryl-protein. The other related compounds were recognized either poorly or not at all. The high affinity and specificity of our monoclonal antibody enabled us to visualize ACh molecules on fixed rat brain sections. ACh was fixed with a mixture of nitrobenzyl alcohol and glutaraldehyde. Many ACh-immunoreactive cell bodies and fibers were seen on sections from the basal forebrain and spinal cord. Preadsorption and other immunohistochemical tests demonstrated that the ACh staining was highly specific.