The effect of insulin and epidermal growth factor on the phosphorylation of CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) was investigated in HeLa cells. For the first time, cytidylyltransferase phosphorylation was shown to be influenced by growth factors in cell culture experiments. The rephosphorylation of cytidylyltransferase after an oleate-mediated dephosphorylation and translocation to membranes was increased after 2 min in the presence of insulin or epidermal growth factor by 99% and 76%, respectively, compared with controls. However, the increased phosphorylation of cytidylyltransferase did not have an effect on its subcellular distribution. Furthermore, purified cytidylyltransferase preincubated with alkaline phosphatase is a substrate for p44mapk, a member of the mitogen-activated protein (MAP) kinase family downstream of the growth factor receptors, in vitro. In accordance with the in vivo data, in vitro phosphorylation of cytidylyltransferase by p44mapk occurred after 2 min.