Insulin exhibited some cross-reactivity with the isolated S-sulfo A- and B-chains in the passive cutaneous anaphylaxis test. The synthetic peptides A1–9, A10–21, and A1–21 gave a passive cutaneous anaphylactic reaction with guinea pig antiserum to natural A-chain, whereas high levels of A10–21 and A1–21 could also inhibit a subsequent insulin challenge from reacting with antiserum to insulin. The synthetic peptides B1–8, B9–30, B24–30, and B1–30 reacted with antiserum to natural B-chain but high levels of peptides (B2–8)2, B9–14, and (B17–23)2 were inactive. The symmetrical double peptide (B1–8)2 reacted with guinea pig antiserum to intact insulin as well as with antiserum to B-chain and certain sera from insulin-resistant diabetic patients. The results indicated that injection of ox insulin into guinea pigs caused production of anaphylactic antibodies mainly towards antigenic loci in the regions A10–21 and B1–8 of the insulin molecule, whereas antigenic determinants in the regions A1–9 and B24–30 were less important.