We have evaluated, by use of isolated canine hepatocytes, the importance of intramolecular hormone cross-linking (and of concomitant changes in molecular flexibility) to the interaction of insulin with its plasma membrane receptor. Cross-linked hormone analogs were prepared by reacting porcine insulin, N alpha A1-t-butyloxycarbonyl insulin or N alpha A1-t-butyloxycarbonyl [D-LysA1]insulin with various dicarboxylic acid active esters to obtain alpha-GlyA1/epsilon-LysB29-, alpha-PheB1/epsilon-LysB29-, and epsilon-D-LysA1/epsilon-LysB29-cross-linked insulins, respectively. In the aggregate, insulin analogs cross-linked by groups containing 2-12 atoms retained 1.4-35% of the receptor binding potency of native insulin. Analysis of our results suggests that: (a) loss of chemical functionality, steric interference, and restriction of potential intramolecular movement can all play roles in determining the receptor binding potencies of cross-linked insulin analogs; (b) restriction of intramolecular movement between residues A1 and B29 affects negatively the binding of insulin to its receptor (but accounts for only a fraction of the conformational change which insulin must undergo to achieve a high affinity state of ligand-receptor interaction); and (c) introduction of a cross-link between residues B1 and B29 (residues that are in fact in proximity in one crystalline form of the hormone) decreases markedly the receptor binding potencies of the corresponding analogs. The importance of these findings is discussed in relation to the potential structure of insulin when it is bound to its plasma membrane receptor.