The buccalin-related peptides, buccalin A and buccalin B, are members of a family of cotransmitters that modulate neuromuscular transmission in Aplysia. In this study, a third buccalin-related peptide, buccalin C, was purified from neuronal elements in the accessory radula closer, a muscle involved in the animal's feeding behavior. Oligonucleotide probes based upon the amino acid sequence of buccalin C were used to isolate cDNA clones that encode a buccalin precursor polypeptide. The buccalin precursor contains 19 distinct buccalin-related peptides, several of which are present in multiple copies. The buccalin gene appears to be present in a single copy, with one allele containing a small insert. Expression of this gene occurs in a tissue-specific manner and mRNA transcripts are abundant within neurons in the Aplysia CNS. This large family of neuropeptides may exert extraordinarily complex modulatory actions at synapses where they serve as cotransmitters.