a-Conotoxins are small disulfide-constrained peptides from cone snails that act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). The 13-residue peptide a-conotoxin RgIA (a-RgIA) is a member of the a-4,3 family of a-conotoxins and selectively blocks the a9a10 nAChR subtype, in contrast to another well-characterized member of this family, a-conotoxin ImI (a-ImI), which is a potent inhibitor of the a7 and a3?2 nAChR subtypes. In this study, we have altered side chains in both the four-residue and the three-residue loops of a-RgIA, and have modified its C-terminus. The effects of these changes on activity against a9a10 and a7 nAChRs were measured; the solution structures of a-RgIA and its Y10W, D5E, and P6V analogues were determined from NMR data; and resonance assignments were made for a-RgIA [R9A]. The structures for a-RgIA and its three analogues were well defined, except at the chain termini. Comparison of these structures with reported structures of a-ImI reveals a common two-loop backbone architecture within the a-4,3 family, but with variations in side-chain solvent accessibility and orientation. Asp5, Pro6, and Arg7 in loop 1 are critical for blockade of both the a9a10 and the a7 subtypes. In loop 2, a-RgIA [Y10W] had activity near that of wild-type a-RgIA, with high potency for a9a10 and low potency for a7, and had a structure similar to that of wild type. By contrast, Arg9 in loop 2 is critical for specific binding to the a9a10 subtype, probably because it is larger and more solvent accessible than Ala9 in a-ImI. Our findings contribute to a better understanding of the molecular basis for antagonism of the a9a10 nAChR subtype, which is a target for the development of analgesics for the treatment of chronic neuropathic pain.