T-cell DNA synthesis and T-helper cell function in response to isolated insulin chains and naturally occurring insulin variants was assessed in insulin immune guinea pigs. Two distinct antigenic determinants, recognized by T cells, were defined. One localized in the B chain and the other one constituted by amino acids A8, A9, and A10 of the insulin A-chain loop. Recognition of the B-chain determinant is under the control of Ir genes linked to the strain 13 major histocompatibility complex. This was shown by studying the response to isolated insulin B chain in F1(2 x 13) guinea pigs, as well as serologically defined backcrosses and outbred animals. Insulin recognition through the A-chain loop determinant is specific for strain 2 guinea pigs. These animals recognize this region of the molecule even when displaying different amino acid sequences. The strain differences observed in those antigenic sites eliciting T-cell recognition was not found at an antibody level. No differences could be detected in the ability of the different insulin variants to inhibit the binding of 125I-labeled pork insulin to strain 2 guinea pig anti-pork insulin or to strain 13 guinea pig anti-pork insulin.