We analyzed the antigenic specificities of T cell hybridomas from normal BALB/c mice and transgenic (Tg) BALB/c mice that express the human insulin gene. Normal mice produce antibodies in response to injection with human insulin (Hins), while Tg mice are tolerant. However, CD4+ T cells can be isolated from Tg mice that proliferate to Hins in vitro. T cells from normal mice were highly specific, recognizing pork insulin (Pins, only 1 aa difference from Hins) but not beef insulin (Bins). The fine specificity of all normal hybridomas tested mapped to the A chain loop (A1-14), and none reacted to the B chain. In contrast, T cells from Tg mice cross-reacted with Pins and Bins. While some Tg T cells responded to the A chain loop, others recognized A chain epitopes outside of the loop region. Unexpectedly, one hybridoma reacted with both the A chain and the B chain, which have no obvious homology. Studies are underway to determine whether the unexpected, dual specificity results from interaction of one TCR with two dissimilar peptides or whether these cells express two TCR each resetiing with a different peptide. We conclude that normal and Tg T cells tend to have different peptide specificites. These differences may provide a basis to study mechanisms of self-tolerance at the molecular level.