omega-Phonetoxin IIA (omegaPtxIIA), a peptide from spider venom (Phoneutria nigriventer), inhibits high threshold voltage-dependent calcium currents in neurons. To define its pharmacological specificity, we have used patch-clamp methods in cell lines expressing recombinant Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 channels (P/Q-, N-, and R-type currents, respectively). Calcium currents generated by Ca(v)2.1 and Ca(v)2.2 were blocked almost irreversibly by 3 nm omegaPtxIIA, whereas Ca(v)2.3 showed partial and readily reversible inhibition. Binding assays with mono[(125)I]iodo-omegaPtxIIA indicated that membranes expressing recombinant Ca(v)2.1 or Ca(v)2.2 channels showed a single class of sites with similar affinity (K(D) approximately 50 pm), whereas low affinity interactions were detectable with Ca(v)2.3. Kinetic, saturation, and displacement assays demonstrated that rat brain synaptosomes displayed multiple classes of binding sites for (125)I-omegaPtxIIA. High affinity binding of (125)I-omegaPtxIIA was totally displaced by omegaPtxIIA (K(i) = 100 pm), but only partially by omega-conotoxin GVIA (25% inhibition) and omega-conotoxin MVIIC (50% inhibition at 0.3 microm). (125)I-omegaPtxIIA thus defines a unique high affinity binding site that is predominantly associated with Ca(v)2.1 or Ca(v)2.2 channels.