The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) from venom of the funnel web spider Agelenopsis aperta is the only known agent that blocks L-type and N-type Ca channels with equal high potency (IC50 < or = 1 nM). From the same venom, we have purified and sequenced a family of peptides which are homologous to omega-Aga-IIIA but vary over 100-fold in their relative affinity for L-type versus N-type Ca channels. One of these, omega-Aga-IIIB, is 76 amino acids long and identical to omega-Aga-IIIA in 66 positions. We identified two other similar peptides, omega-Aga-IIIC and omega-Aga-IIID, as well as one single amino acid variant of omega-Aga-IIIA and two of omega-Aga-IIIB. The type III omega-agatoxins exhibit similar but distinct activities on voltage-gated Ca channels. omega-Aga-IIIA, omega-Aga-IIIB, and omega-Aga-IIID are nearly indistinguishable in their actions at the insect neuromuscular junction (no effect at 0.1 microM), on atrial T-type Ca channels (no effect at 0.5 microM), and in two assays for synaptosomal Ca channels: they are nearly equipotent inhibitors of 125I-omega-conotoxin GVIA binding to rat brain synaptic membranes (IC50 = 0.17-0.33 nM) and blockers of the K(+)-induced 45Ca2+ influx into chick brain synaptosomes (omega-Aga-IIIB, 1.2 nM; omega-Aga-IIIA, 2.4 nM). In contrast, omega-Aga-IIIA is a better blocker of locust Ca channels (IC50 approximately 10-50 nM) than is omega-Aga-IIIB. Finally, although omega-Aga-IIIA, omega-Aga-IIIB, and omega-Aga-IIID all block atrial L-type Ca channels, omega-Aga-IIIA is over 100-fold more potent. Thus, although type III omega-agatoxins appear to recognize a binding site common to L- and N-type Ca channels, omega-Aga-IIIB and omega-Aga-IIID identify differences between the two channels.