Cyclopentenone prostaglandins (PGs), particularly those of the J-series, affect proliferation and differentiation in a number of cell lines. J-ring PGs have been shown to be ligands for the peroxisome proliferator-activated receptor (PPAR)-γ and to modulate NF-κB-mediated gene transcription. We have previously reported that large quantities of eicosanoids, including PGJ₂, are produced by the human colorectal cancer cell line HCA-7 while lesser amounts of Δ¹²-PGJ₂ and 15-deoxy-Δ^12,14-PGJ₂ are formed. In this and other cell lines, cyclopentenone PGs have been shown to increase cell proliferation, but factors that influence their formation and metabolism are poorly understood. Unlike other PGs, cyclopentenone PGs contain α,β-unsaturated carbonyl groups that readily adduct various biomolecules such as glutathione (GSH) in vitro. We now report that in HCA-7 cells, PGJ₂ is largely metabolized by conjugation to GSH. Characterization of the adducts by liquid chromatography (LC)–mass spectrometry (MS) revealed two major metabolites consisting of (1) a novel GSH conjugate in which the carbonyl at C-11 of PGJ₂ is reduced and (2) intact PGJ₂ conjugated to GSH. Approximately 70% of the PGJ₂ added to HCA-7 cells was esterifed to GSH after 2 h of incubation, suggesting this pathway represents the major route of metabolic disposition of PGJ₂ in HCA-7 cells.