Phosphatidylethanolamine (PE) is an essential membrane phospholipid and important supplier of biologically active lipid-derived molecules. The present study includes various aspects of regulation of the CDP-ethanolamine pathway and the rate-controlling enzyme, Pcyt2, in the 'de novo' biosynthesis of PE in human breast cancer cells, MCF-7. Both mouse and human Pcyt2 promoters and human PCYT2 mRNA levels are down-regulated by serum lipids and delipidated serum (serum growth factors), and in contrast significantly up-regulated in the absence of lipids and serum. Fatty acids, oleic acid and EPA added to the delipidated serum media significantly inhibit the PCYT2 promoter and enzyme activity, which is likely driven at the transcriptional level. 25-OH-cholesterol was a strong inhibitor of the PCYT2 promoter-luciferase activity in transient expression experiments. However, the promoter assay did not correspond to the changes in endogenous PCYT2 activity and it is possible that multiple mechanisms are involved in its regulation.