The effect of in vivo treatment with THF (thymus humoral factor) on the response of mouse lymphocytes to mitogens was investigated. Different preparations of THF were tested by daily i.m. injections into normal mice. Spleen, peripheral lymph node and thymus cells were tested for reactivity to several mitogens at various intervals after initiation of treatment. Our results indicate that the administration of THF to normal mice causes an inhibitory trend in the response of spleen cells to T-cell mitogens. It was found that the response of spleen cells to phytohemagglutinin (PHA) and concanavalin A (Con A) was strongly depressed after 7 days of treatment with 50 micrograms of THF. This was followed by a recovery of the response to normal levels after 14 days of treatment. The response of spleen cells to lipopolysaccharide (LPS) a B-cell mitogen, did not change throughout 14 days of treatmnent with THF. Lymph node cells of THF-treated mice behaved qualitatively in a similar manner to spleen cells, while thymus cells from THF-treated mice showed a significantly increased response to Con A. In contrast to normal mice, the injection of THF into mice with an impaired reactivity to PHA, such as thymectomized mice and tumor-bearing mice, caused an augmentation of the mitogenic response of spleen cells. These results suggest that in vivo administration of THF causes regulatory changes in the lymphocyte reactivity to T-cell mitogens.