A new peptide antagonist of voltage-activated calcium channels was purified from venom of the funnel web spider, Agelenopsis aperta. This 48-amino acid peptide, omega-agatoxin (omega-Aga)-IVB, was found to be a potent (Kd, approximately 3 nM) blocker of P-type calcium channels in rat cerebellar Purkinje neurons but had no activity against T-type, L-type, or N-type calcium channels in a variety of neurons. The calcium channel-blocking properties of omega-Aga-IVB were similar to those of another toxin, omega-Aga-IVA, which has 71% amino acid identity with omega-Aga-IVB. The 10-fold greater abundance of omega-Aga-IVB in venom allowed structural studies using NMR spectroscopy. The three-dimensional structure derived from NMR data resulted in a proposed disulfide bond configuration for the peptide. Although omega-Aga-IVB has fewer basic and more acidic residues than does omega-Aga-IVA, the two toxins show conservation of positively charged residues in a mid-peptide region that is predicted to form one face of the omega-Aga-IVB molecule. This region may be crucial for high affinity binding to the P-type calcium channel. In contrast, the amino termini of the two toxins have different charges and seem unlikely to be involved in binding to the channel.