T lymphocyte responses to heterologous or self 65-kD heat shock protein (hsp) have been implicated in the pathogenesis of various forms of arthritis. To delineate the relationship of 65-kD hsp to different synovial fluid (SF) T cell subsets, we stimulated synovial fluid (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with different inflammatory rheumatic diseases and from healthy controls with human or mycobacterial 65-kD hsp, tetanus toxoid (TT), heat-killed or live Yersinia enterocotitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR-γδ⁺ lymphocytes in the 65-kD hsp-stimulatcd SF-derived lines. This expansion of TCR-γδ⁺ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR-γδ⁺ cells was also shown after SFMC stimulation with live, but not with heat-killed Yersinia or with TT. We conclude that a common mechanism is involved in the selective expansion of TCR-γδ⁺ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65-kD hsp. Out of a panel of TCR-γδ⁺ T lymphocyte clones (TLC) derived from a human 65-kD hsp-stimulated line, only a minority of TLC proliferated weakly upon restimulation with this antigen in the presence of autologous monocytes, whereas TCR-αβ⁺ TLC responded vigorously to the human 65-kD hsp and in some cases also cross-recognized the mycobacterial hsp homologue and/or heat-killed Yersinia. This implies that additional factors or cells may be present in the milieu of SFMC cultures that propagate the expansion of TCR-γδ⁺ cells in response to 65-kD hsp or live bacteria.