The significance of phosphatidylethanolamine (PE) in breast cancer cell metabolism was investigated under stress conditions caused by serum deficiency. Serum deficient MCF-7 cells adapt to stress conditions by increasing synthesis and content of PE and diacylglycerol (DAG). The biosynthesis of PE from DAG and ethanolamine was regulated at the level of formation of CDP-ethanolamine, the metabolic step catalyzed by Pcyt2. The catalytic activity of Pcyt2 was elevated 2-3-fold, yet the enzyme remained rate-limiting in serum-deficient cells. Contributions to the elevated Pcyt2 activity included transcriptional and translational components. The mRNA levels of two splice variants, Pcyt2α and Pcyt2β, were 1.5-3-fold higher in deficient cells. The total amounts of Pcyt2 and Pcyt2α proteins were similarly elevated 1.5-2.5-fold. In vivo [γ(32)Pi] radiolabeling revealed that Pcyt2 was additionally regulated by phosphorylation. Under unfavorable metabolic conditions, both endogenous and His/Myc-tagged Pcyt2 were increasingly phosphorylated at Ser residues. The results established that elevated DAG formation and the increased activity of the rate-regulatory enzyme Pcyt2 were critical modulators of the PE Kennedy pathway, and total PE content in serum deprived breast cancer cells. Therefore, as an essential gene sensitive to nutritional microenvironment, Pcyt2 could represent a legitimate target in novel metabolic strategies for cancer.