The receptor-binding surface of insulin is broadly conserved, reflecting its evolutionary optimization. Neighboring positions nevertheless offer an opportunity to enhance activity, through either transmitted structural changes or introduction of novel contacts. Nonconserved residue A8 is of particular interest as Thr(A8) --> His substitution (a species variant in birds and fish) augments the potency of human insulin. Diverse A8 substitutions are well tolerated, suggesting that the hormone-receptor interface is not tightly packed at this site. To resolve whether enhanced activity is directly or indirectly mediated by the variant A8 side chain, we have determined the crystal structure of His(A8)-insulin and investigated the photo-cross-linking properties of an A8 analogue containing p-azidophenylalanine. The structure, characterized as a T(3)R(3)(f) zinc hexamer at 1.8 A resolution, is essentially identical to that of native insulin. The photoactivatable analogue exhibits efficient cross-linking to the insulin receptor. The site of cross-linking lies within a 14 kDa C-terminal domain of the alpha-subunit. This contact, to our knowledge the first to be demonstrated from the A chain, is inconsistent with a recent model of the hormone-receptor complex derived from electron microscopy. Optimizing the binding interaction of a nonconserved side chain on the surface of insulin may thus enhance its activity.