The highly conserved 60kD endogenous heat shock protein (hsp60) has been suggested to be a target for T cell recognition in autoimmune diseases such as type I diabetes. We previously reported cross-recognition of both mycobacterial hsp60 (Mt60) and self hsp60 (m60) by Mt60 immunized NOD mice. To identify the epitopes involved, we generated T cell lines against m60 or its mycobacterial counterpart and tested these lines for recognition of complete sets of overlapping peptides spanning either hsp60 sequence. T cell lines responded to identical regions in the hsp60 proteins, regardless of their degree of conservation or I-Ag7 binding-affinity. Additionally, we determined whether a protective genetic background would affect the presence of hsp60 cross-reactive T cells in the peripheral repertoire by comparing epitope recognition in I-Ag7 expressing NOD, NOD-asp and Biozzi AB/H mice. Two out of five immunodominant murine peptides were able to induce proliferation in NOD and NOD-asp Mt60 T cell lines, but not in Biozzi AB/H T cell lines. Our results point out that Mt60 immunization not necessarily leads to proliferative T cells responding to endogenous hsp60 peptides in the context of diabetes-predisposing I-Ag7. Moreover, the capacity of T cells to respond to self hsp60 is not influenced by the presence of protective I-Ag7asp. Yet, proliferation of hsp60 autoreactive T cells is solely measured in combination with insulitis and as such serves as a surrogate marker for islet inflammation.