Synthesis of phosphatidylcholine, the major phospholipid of animal cell membranes, requires the key enzyme cytidylyltransferase (CCTalpha). Cysteine sulfhydryls within CCTalpha are needed for full catalytic activity. Here we show that prostaglandin 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) inactivates CCTalpha by inducing generation of reactive oxidant species and the appearance of a cross-linked CCTalpha dimer in cells. N-Acetyl-l-cysteine reduced oxidative stress, prevented CCTalpha cross-linking, and restored CCT function in 15d-PGJ2-treated cells. 15d-PGJ2 modified critical cysteine residues within CCTalpha as determined by mutagenesis studies and by incorporation of biotin-15d-PGJ2 into CCTalpha. These effects of 15d-PGJ2 were associated with CCTalpha accumulation within the nucleus. The data indicate that bioactive prostanoids significantly impair membrane phospholipid production by promoting cysteine cross-bridging within CCTalpha.