In the course of purification of omega-Aga-IVA, a specific P-type calcium channel blocker, from the venom of Agelenopsis aperta we discovered a novel peptide. This peptide, named omega-agatoxin Tsukuba (omega-Aga-TK), also blocked P-type channels and was twelve times more abundant in the venom than omega-Aga-IVA. omega-Aga-TK was purified to homogeneity by a two-step reverse-phase HPLC procedure. Its amino acid sequence is 71% identical to that of omega-Aga-IVA. omega-Aga-TK has a negatively charged N-terminus, whereas omega-Aga-IVA has a positively charged one. Electrophysiological data indicate that omega-Aga-TK is a potent and selective inhibitor of P-type channels.