Cell-cycle progression of rat thymocytes stimulated with concanavalin A and interleukin 2 was monitored at 12-h intervals by pulse labeling aliquots of the cell culture with [3H]thymidine, by measuring cellular DNA and protein content and by counting the number of cells in the cultures. The cell cycle was completed after 96 h of culture with the S phase peaking at 48 h. Early events in thymocyte activation were enhanced phosphatidylinositol turnover and the induction of ornithine decarboxylase. Concomitant changes were observed in the rates of DNA synthesis and glycolysis accompanied by a 20-fold increase in glucose uptake 48 h after stimulation. However, the maximal increment in the glycolytic rate preceded that of DNA synthesis by 12 h. Apart from the quantitative changes which occurred during the cell-cycle progression, there was also a change from partial aerobic glucose degradation to COz (26%) to almost complete anaerobic conversion of glucose to lactate (85%) and less than 3% to COz. Glycolytic enzyme levels increased fourfold to tenfold and reached their maxima 48 h after mitogenic stimulation. Maximal increments of glycolytic enzyme activities preceded or coincided with the maximal increments of the glycolytic rate. Actinomycin D (1.5 ng/ml) completely inhibited DNA and RNA synthesis but did not show any inhibitory effect either on glycolytic enzyme induction or on enhanced glycolysis. During mitosis and return of the cells to the non-proliferative state, all of the enhanced metabolic rates returned to their initial levels and the elevated enzyme activities were decreased also. The marked changes of metabolic rates and enzyme activities observed at the various phases of the cell cycle suggest that these biochemical events may also serve as suitable parameters for evaluating the response of lymphocytes towards mitogens and lymphokines.Activation of quiescent rat thymocytes by polyclonal mitogens such as concanavalin A (ConA) involves a complex cascade of biochemical reactions which result in promoting cell-cycle entry and expression of receptors for interleukin 2 (11-2). On addition of 11-2, progression through GI to DNA synthesis in S phase and final proliferation of thymocytes occurs. Recent studies of lymphocyte activation have implicated an increased conversion of phosphatidylinositol into inositol trisphosphate and diacylglycerol already as an early event [l -71. Subsequently the phospholipid/Ca2 '-dependent protein kinase C [8] is activated and intracellular Ca2+ concentration as well as pH [9 -171 increases. Cell-cycle advancement from the resting Go to the activated G I stage also inCorrespondence to K. Brand,