Substantial increases in both 3beta-OH sterol and fatty acid synthesis were observed after concanavalin A addition to mouse spleen lymphocytes cultured in serum-free media. The rate of sterol synthesis increased linearly up to 60 h. The rate of fatty acid synthesis increased up to 20 h, reaching a plateau in synthetic activity which was the maintained. CO2 production from acetate was slightly stimulated by concanavalin A. In contrast to sterol and fatty acid synthesis, the rate of CO2 production in both mitogen-stimulated and resting cultures declined with time. Dibutyryl cyclic AMP had a strong inhibitory effect on concanavalin A-stimulated sterol and fatty acid synthesis from acetate, but only a slight effect on CO2 production. Delayed addition of dibutyryl cyclic AMP resulted in reduced inhibition. The data suggest a sequence of initiation for fatty acid and sterol synthesis prior to DNA synthesis and a possible regulatory role of cyclic AMP in this initiation. The results support the hypothesis that lymphocyte activation is sequential within the spleen cell population and is accompanied by fatty acid and sterol synthesis.