ω-Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new ω-conotoxins (CVIA-D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other ω-conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA-D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, ω-conotoxins CVID and MVIIA had similar potencies to inhibit current through central (α(1B-d)) and peripheral (α(1B-b)) splice variants of the rat N-type calcium channels when coexpressed with rat β3 in Xenopus oocytes. However, the potency of CVID and MVIIA increased when α(1B-d) and α(1B-b) were expressed in the absence of rat β3, an effect most pronounced for CVID at α(1B-d) (up to 540-fold) and least pronounced for MVIIA at α(1B-d) (3-fold). The novel selectivity of CVID may have therapeutic implications. 1H NMR studies reveal that CVID possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.