While the major population of T lymphocytes express T cell receptor (TCR) alpha beta-chains and recognize peptide antigens in association with either Major Histocompatibility Complex class I or class II molecules, a consensus view does not exist concerning either the nature of the antigen recognized or the nature of the restriction element utilized by the minor population of T cells which express TCR gamma delta-chains. We have identified a unique subpopulation of gamma delta T cells which uniformly express the C gamma 4, V delta 6 TCR and which produce a number of cytokines in the absence of exogenous stimulation. Adaption of these cell lines to serum-free culture conditions resulted in a cessation of cytokine production which could then be induced by the addition of extracellular matrix (ECM)-proteins to the culture. The response to the ECM-proteins could be completely inhibited by an antibody to the murine vitronectin receptor (VNR). However, engagement of the VNR by its ligand was not sufficient for the induction of cytokine production as anti-TCR antibodies inhibited the response to ECM-proteins and gamma delta TCR loss mutants failed to respond. Collectively, these data demonstrate that not only is coexpression of the VNR and the gamma delta TCR required for the induction of cytokine production by this subpopulation of T cells, but that the TCR must also be engaged by its ligand, most likely a cell surface autoantigen expressed by the T cells themselves.