Inhibition of calcium-evoked [3H]ACh release by different classes of calcium channel blockers was compared among calcium-naive synaptosomes from chick, frog and rat forebrain tissues. In all three species, [3H]ACh release was insensitive to nifedipine (0.03-3 microM) but was completely inhibited by cadmium (IC50 range = 0.7-1.7 microM) or cobalt (190-350 microM). In contrast, the peptide omega-conotoxin revealed marked species heterogeneity in that [3H]ACh release was potently blocked in chick and rat synaptosomes (IC50 congruent to 1 nM), whereas frog tissue was notably resistant (IC50 greater than 100 nM). Together, these data provide functional evidence for pharmacological heterogeneity among presynaptic calcium channels that subserve [3H]ACh release.