Incubation of peripheral blood or isolated lymphocytes of C57L/J mice with phytohemagglutinin stimulated the incorporation of thymidine into DNA of lymphocytes as they transformed into large lymphoblasts. DNA synthesis began after about 24 hr of incubation and reached a peak at 48 hours. The de-novo synthesis of sterols from acetate was stimulated much earlier, at 4 hr of incubation, and the rate reached a maximum at 24 hr, approximately at the time DNA synthesis began. Rates of incorporation of radioactivity from [14-C]acetate into fatty acids and into CO2 by phytohemagglutinin-treated blood were not significantly different from control values. Phytohemagglutinin stimulation of sterol synthesis could be abolished by the addition of certain oxygenated derivatives of cholesterol (e.g., 25-hydroxycholesterol and 20alpha-hydroxycholesterol) which specifically depress the activity of the regulatory enzyme in the sterol synthesis pathway, 3-hydroxy-3-methylglutaryl CoA reductase [mevalonate:NADP-nOXIDOREDUCTASE (CoA acylating); EC 1.1.1.34]. This treatment also abolished DNA synthesis and blastogenesis which otherwise followed the peak of sterol synthesis. Furthermore, DNA synthesis was repressed only if the inhibitor was added early enough to prevent sterol synthesis from reaching its usual maximum. When the compound was added after the rate of sterol synthesis had reached its maximum, DNA synthesis was not affected. These findings suggest that the synthesis of cholesterol is an essential prerequisite for successful initiation and completion of the cell cycle in lymphocytes after phytohemagglutinin activation.