Omega-Grammotoxin SIA is a peptide isolated from tarantula venom on the basis of its ability to block the voltage-gated Ca2+ channels that mediate glutamate release. To determine the Ca2+ channel subtype selectivity of omega-grammotoxin SIA, whole-cell Ba2+ current (IBa) was measured in cultured rat hippocampal neurons. Selective Ca2+ channel blockers were used to identify components of IBa mediated by Ca2+ channel subtypes. omega-Agatoxin IVA at 30 nM, 1 microM omega-conotoxin GVIA, and 3 microM omega-contoxin MVIIC, applied consecutively, each elicited a fractional increase in the cumulative block of IBa, identifying components of IBa mediated by P-, N-, and Q-type calcium channels. omega-Grammotoxin at 1 microM, a maximally effective concentration, blocked 52% of IBa. omega-Conotoxin MVIIC and the combination of omega-conotoxin GVIA and micromolar omega-agatoxin IVA blocked 52% and 54% of IBa, respectively, and block of IBa by omega-grammotoxin SIA was mutually occlusive of block of IBa by either treatment, both of which block N-, P-, and Q-type Ca2+ channels. The L channel blocker nimodipine produced identical block of IBa in the presence and absence of omega-grammotoxin SIA. These results indicate that omega-grammotoxin SIA blocks N-, P-, and Q-type but not L-type voltage-gated calcium channels. Block of IBa by omega-grammotoxin SIA was faster in onset and less sensitive to external divalent cation concentrations than was block by omega-conotoxin MVIIC, and it was rapidly and substantially reversible. Rapid onset, relative insensitivity to divalent cation concentrations, and reversibility render omega-grammotoxin SIA a useful tool for inhibition of neuronal voltage-gated Ca2+ channels.