Poliovirus increases phosphatidylcholine biosynthesis in HeLa cells by stimulation of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase (Vance, D.E., Trip, E.M., and Paddon, H.B. (1980) J. Biol. Chem. 255, 1064-1069). The mechanism for the virus effect has been investigated. An assay for the cytidylyltransferase which mimics the physiological conditions within the cell was developed. The enzyme activity was not changed at 3 h but was stimulated more than 2-fold at 4 and 5 h after infection with poliovirus. Enzyme activity was stimulated by addition of CTP to the assay. At 0.10 mM CTP the difference in activities from poliovirus- and mock-infected cells was abolished. Mg2+ inhibited the cytidylyltransferase activities and eliminated the differences between the two activities at a concentration of 0.05 mM. However, the endogenous amount of Mg2+ in the postmitochondrial supernatants was the same for infected and mock-infected cells. The addition of CDP-choline or PPi inhibited the cytidylyltransferase activity but had no effect on the relative differences in activities from infected and mock-infected cells. Measurement of CTP in the postmitochondrial fraction showed no differences at 3 h but was elevated 2- to 3-fold in poliovirus-infected cells at 4 and 5 h. It appears that the cytidylyltransferase reaction is faster in poliovirus-infected HeLa cells because of an increase of CTP in the cytoplasmic compartment. Moreover, it appears that the concentration of CTP in the cytoplasm can determine the rate of phosphatidylcholine biosynthesis in HeLa cells.