Previous studies have demonstrated that 12-O-tetradecanoyl phorbol 13-acetate (TPA) stimulates phosphatidylcholine biosynthesis in HeLa cells. The stimulation was apparently caused by an acceleration of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15) (Paddon, H.B. and Vance, D.E. (1980) Biochim. Biophys. Acta 620, 636-640). We now provide evidence that the enzyme activation is due to a translocation of the cytidylyltransferase from the cytosol to the microsomes. The rate of phospho[Me-3H]choline conversion into phosphatidylcholine was approx. 3-fold faster in HeLa cells treated with 100 nM TPA. This stimulation correlated with a 2.3-fold activation (P less than 0.05) of cytidylyltransferase in homogenates from treated cells. There was a 1.7-fold increase in the enzyme associated with microsomes (P less than 0.05) and a corresponding decrease in enzyme recovered from cytosol (P less than 0.01). The total amount of enzyme recovered from the homogenates was unchanged. Further evidence for TPA causing an increased association of cytidylyltransferase with cellular membranes was obtained when cells were treated with digitonin. The release of cytidylyltransferase into the medium was inhibited by 4-fold from cells previously treated with TPA. Similar results on phospho[Me-3H]choline incorporation into phosphatidylcholine were found with cells incubated with phorbol-12,13-dibutyrate, a water-soluble tumor-promoting agent.