The regulation of choline kinase activity by fetal bovine serum and the regulation of phosphatidylcholine biosynthesis by choline kinase have been investigated in 3T3 fibroblasts. Treatment of quiescent 3T3 fibroblasts with serum was shown in previous work to increase phosphocholine pool size and phosphatidylcholine biosynthesis. We now report that treatment of 3T3 cells with serum increased intracellular choline kinase activity by 2-3-fold with a concomitant 2-3-fold decrease of intracellular free choline concentrations. Initial rates of choline transport were the same in quiescent and serum-treated cells, whereas choline kinase activity was 2-3-fold higher in serum-treated cells. As a consequence, free choline concentrations were 2-3-fold lower in serum-stimulated cells than in control quiescent cells. Phosphocholine turnover rates were increased 2-fold by serum treatment both as a consequence of a serum-dependent increase of phosphocholine pools and as a result of a serum-dependent lowering of the phosphocholine half-life. Thus, the overall response of 3T3 cells to serum stimulation included decreased choline pools and increased choline kinase activity, phosphocholine pool size, phosphocholine turnover, and phosphatidylcholine biosynthesis.