Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6-A11, A7-B7, and A20-B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7-A11, A6-B7, and A20-B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His(B5) --> Thr markedly destabilizes the hormone (DeltaDeltaG(u) 2.0 +/- 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution Thr(B5) --> His (residue 4) specifies a unique structure with native (1)H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, His(B5)-IGF-I retains canonical pairing. Chemical denaturation studies indicate that His(B5) does not significantly enhance thermodynamic stability (DeltaDeltaG(u) 0.2 +/- 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of Thr(B5)-insulin is decreased 5-fold, His(B5)-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of Thr(B5) in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of His(B5) in insulin highlights its critical role in insulin biosynthesis.