We have shown previously that atherosclerotic lesions can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein 65 (hsp65), which has a high degree of sequence homology with mammalian hsp60. To investigate a possible relationship between hsp60 expression and the antigenic specificities of infiltrating T cells in the lesion, 38 New Zealand White rabbits were treated either by immunization with recombinant mycobacterial hsp65 or by administration of a 0.2% cholesterol diet. Atherosclerotic lesions were observed after 16 wk, particularly in the aortic arch and arterial bifurcations of rabbits immunized with hsp65 or fed with a cholesterol-rich diet. Hsp65 staining of aortas showed a heterogeneous distribution, and significantly increased staining intensity in atherosclerotic lesions compared to aortic media or adventitia. This abundantly expressed hsp65 was observed in atherosclerotic lesions induced by hsp65 immunization as well as those induced by cholesterol-rich diet alone. Interestingly, a population of the T lymphocytes isolated from all forms of atherosclerotic lesions specifically responded to hsp65 in vitro. IL-2-expanded T cell lines derived from atherosclerotic lesions showed a significantly higher hsp65 reactivity than those developed from peripheral blood of the same donor. Furthermore, levels of circulating antibodies and numbers of spleen cells specifically reacting against hsp65 were elevated in all experimental animals. Flow cytometric analysis of spleen cells showed elevated immune response-associated antigen expression in treated animals. In conclusion, increased hsp65 expression in intimal cells and the presence of hsp65-specific T cells in blood and in atherosclerotic lesions may be important in initiating the development of atherosclerosis and perpetuating the lesions.