We investigated block of voltage-dependent Ca channels in freshly dissociated rat central and peripheral neurons by the synthetic peptide omega-Aga-IVA. Synthetic omega-Aga-IVA blocked approximately 90% of the high-threshold Ca current in cerebellar Purkinje neurons with an estimated Kd of approximately 1.5 nM, slightly higher than that determined for block by toxin purified from Agelenopsis aperta venom. At 200 nM, the synthetic peptide blocked a small fraction of current in dorsal root ganglion neurons but had no effect on identified components of current carried by low-threshold T-type channels or by dihydropyridine-sensitive L-type Ca channels. Up to 800 nM synthetic peptide had no effect on the current in sympathetic neurons, carried mainly by omega-conotoxin GVIA-sensitive N-type channels. In spinal cord neurons, the same fraction of high-threshold current was blocked by synthetic peptide or purified toxin. We conclude that synthetic omega-Aga-IVA has the same high selectivity for blocking P-type Ca channels as does toxin purified from spider venom.