In the present study we evaluated the effect of sex hormone on the generation of murine cytotoxic T cell responses. We show that the in vitro CTL response was strongly inhibited by progesterone but not by E1, E2 or testosterone. Our experiments attempting to understand the mechanism of the hormone action on CTL development have revealed that the ability of the cells to generate helper signals was not affected. This was demonstrated by the fact that neither IL-2 production nor IL-2 receptor expression was altered by the hormone. Rather, it appears that the capacity of the cells to respond to the signals and to become cytotoxic was modified. Furthermore, we show that the hormone mediated an inhibition of CTL development in thymocyte cultures externally supplemented with all the required helper factors. These results strongly suggest that progesterone has a direct effect on the differentiation of cytotoxic effector cells.