Interactions between gamma delta T cells and heat shock proteins (HSP) have been proposed as contributing factors in a number of diseases of possible autoimmune etiology but definitive evidence to support this hypothesis has been lacking. In multiple sclerosis (MS), a chronic inflammatory neurologic disease, HSP and gamma delta T cells are known to colocalize in brain lesions. Analysis of T cell receptor (TCR) gene usage in these lesions has detected evidence of clonality within both the V delta 2-J delta 1 and V delta 2-J delta 3 populations of gamma delta T cells. In our own studies, using direct sequence analysis, a dominant V delta 2-J delta 3 TCR sequence was found in 9 MS brain samples, suggesting a response to a common antigen. In this report, we have examined gamma delta T cell receptor gene usage in MS peripheral blood T cell lines selected for reactivity to HSP 70. TCR rearrangement patterns for V delta 2-J delta 1 and V delta 2-J delta 3 were studied using the polymerase chain reaction (PCR) and a direct sequencing technique in populations of peripheral blood mononuclear cells (PBMC) cultured with Mycobacterium tuberculosis (M. tuberculosis) purified protein derivative (PPD) and then selected for reactivity to a 70-kD heat shock protein (HSP70). Cells were obtained from health donors, patients with MS, and patients with tuberculosis (TB). PCR products were subjected to direct sequence analysis to look for evidence for clonality within these T cell lines and to define the sequence of the V-D-J (CDR3) region of the TCR. In freshly isolated PBMC, both V delta 2-J delta 1 and V delta 2-J delta 3 gene rearrangement patterns were detected, whereas in HSP70+ T cell lines the predominant delta chain rearrangement pattern was V delta 2-J delta 3. Direct sequence analyses indicated that in cells reactive with HSP70 the V delta 2-J delta 3 sequences were usually oligoclonal and used D delta 3 exclusively. In four of four MS and two of three TB patients, the oligoclonal sequences in the HSP70+ T cell lines were identical to one another and to a dominant sequence previously detected in MS brain lesions. In two of three HSP70+ T cell lines from healthy controls, the oligoclonal sequences differed from those found in both groups of patients but were identical to one another except for a small region of heterogeneity in the second N region. In contrast, in freshly isolated PBMC or in PPD+HSP70- T cell lines, the V delta 2-J delta 3 gene rearrangement patterns were usually polyclonal and dominant sequences were rarely identified. These results support the conclusion that a subpopulation of gamma delta T cells in MS lesions are responding to HSP 70 and that non-CNS-specific antigens contribute to the pathogenesis of MS.