Abstract Actions of ω-conotoxin GVIA on synaptosomes isolated from a Japanese electric ray, Narke japonica, were investigated. ω-Conotoxin inhibited, in a dose-dependent manner, both increases in free calcium concentration in, and acetylcholine release from synaptosomes depolarized with a high concentration of potassium ions. The concentrations of ω-conotoxin required for half-maximal inhibition ( ic 50) of increase in intrasynaptosomal Ca and acetylcholine release were 8 and 7 μM, respectively. Assay using radioiodinated toxin derivative revealed a specific binding site with a dissociation constant ( K D) of 2.8 μM and a density ( B max) of 45 pmol/mg protein of synaptosome. Binding assay with synaptosomal plasma membrane showed a K D = 7μ M and a B max = 200pmol/mg protein. Autoradiography with sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after covalent cross-linking of the toxin, using disuccinimidyl suberate, revealed the 170,000 mol. wt peptide to be an ω-conotoxin receptor. The present study has directly and clearly shown that ω-conotoxin inhibits acetylcholine release by blocking Ca influx into nerve terminals. The 170,000 mol. wt peptide identified as a receptor of the toxin exists in high density in the plasma membrane of the presynaptic nerve terminal and is likely to be a component of a voltage-dependent Ca channel responsible for the neurotransmitter release.