The lymphocyte response to endotoxin (LPS) has been attributed largely to the action of this agent as a polyclonal activator of B lymphocytes. In this study we found that a cloned murine interleukin 2-dependent cytotoxic T cell line, CT 6, proliferates in response to LPS, thus providing the first evidence that T cells can be stimulated directly by LPS. The response was dose and time dependent and was blocked by polymyxin B, an inhibitor of LPS-induced mitogenesis. The fact that this is a cloned T cell line, free of other potentially contaminating lymphoid cell types, precludes the possibility that this proliferation is due to contaminating B lymphocytes or is mediated by macrophage-derived products such as interleukin 1. Moreover, highly purified splenic T lymphocyte populations (purified by negative/positive selection or by a rigorous column purification procedure) contain a small subpopulation (approximately 3%) of T cells that proliferate in response to LPS. This population is missing in the endotoxin-hyporesponsive C3H/HeJ mouse. As was observed in the CT 6 line, proliferation of splenic T cells in response to LPS was inhibited by polymyxin B. Furthermore, treatment of LPS-stimulated T cells with anti-T cell antibodies plus complement blocks the uptake of 3H-thymidine by these cultures. Exogenous interleukin 1 failed to stimulate the T cell cultures comparably to LPS and therefore cannot account for the degree of stimulation observed. These findings support and extend previous findings that suggested a role for an endotoxin-sensitive T cell population in the induction of certain responses, such as LPS-induced adjuvanticity of the lymphocyte-dependent LPS induction of macrophage procoagulant activity.