The effect of the thymus humoral factor (THF) on the response of different lymphoid cell populations to T mitogens was studied. Lower THF concentrations were required to increase the response to concanavalin A (Con A) than to increase that to phytohemagglutinin (PHA), suggesting that T cell types at various stages of maturation are activated at different THF concentrations. Lower THF concentrations were required to enhance the mitogenic response of thymus cells than to enhance the mitogenic response of spleen cells, and this may be because of the higher incidence of target cells for THF activity in the thymus cell population. Spleen cells of adult thymectomized mice exhibited a higher sensitivity for THF than cells of intact control mice. Lymph node cells showed no increase in motogenic response after THF treatment. Similarly, THF did not affect the mitogenic response of spleen cells from nude mice. These results confirm our hypothesis that the targets for THF activity are the younger cells within the T cell lineage.