Synthesis of phosphatidylcholine, the major phospholipid of animal cell membranes, requires the key enzyme cytidylyltransferase (CCTα). Cysteine sulfhydryls within CCTα are needed for full catalytic activity. Here we show that prostaglandin 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) inactivates CCTα by inducing generation of reactive oxidant species and the appearance of a cross-linked CCTα dimer in cells. N-Acetyl-L-cysteine reduced oxidative stress, prevented CCTα cross-linking, and restored CCT function in 15d-PGJ2-treated cells. 15d-PGJ2 modified critical cysteine residues within CCTα as determined by mutagenesis studies and by incorporation of biotin-15d-PGJ 2 into CCTα. These effects of 15d-PGJ2 were associated with CCTα accumulation within the nucleus. The data indicate that bioactive prostanoids significantly impair membrane phospholipid production by promoting cysteine cross-bridging within CCTα.