The peptides isolated from venoms of predatory marine Conus snails ("conotoxins") are well-known to be highly potent and selective pharmacological agents for voltage-gated ion channels and receptors. We report the discovery of two novel TTX-resistant sodium channel blockers, mu-conotoxins SIIIA and KIIIA, from two species of cone snails. The two toxins were identified and characterized by combining molecular techniques and chemical synthesis. Both peptides inhibit TTX-resistant sodium currents in neurons of frog sympathetic and dorsal root ganglia but poorly block action potentials in frog skeletal muscle, which are mediated by TTX-sensitive sodium channels. The amino acid sequences in the C-terminal region of the two peptides and of the previously characterized mu-conotoxin SmIIIA (which also blocks TTX-resistant channels) are similar, but the three peptides differ in the length of their first N-terminal loop. We used molecular dynamics simulations to analyze how altering the number of residues in the first loop affects the overall structure of mu-conotoxins. Our results suggest that the naturally occurring truncations do not affect the conformation of the C-terminal loops. Taken together, structural and functional differences among mu-conotoxins SmIIIA, SIIIA, and KIIIA offer a unique insight into the "evolutionary engineering" of conotoxin activity.