An unusual cyclooxygenase-derived metabolite of arachidonic acid has been shown to be produced by N,N-dimethylformamide (DMF)-induced, terminally differentiated human HL-60 promyelocytic leukemia cells and to a much lesser extent by untreated cells. Biochemical evidence in conjunction with gas chromatography/mass spectrometry and liquid chromatography/thermospray mass spectrometry analyses indicates that the product is 12-keto-5,8,10-heptadecatrienoic acid (KHT). Both KHT and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) were produced when arachidonic acid was incubated with cell lysates obtained from differentiated HL-60 granulocytes. Indomethacin and the thromboxane synthetase inhibitor UK-38485 inhibited the production of both metabolites, whereas ethacrynic acid inhibited only the production of KHT. In 100,000 g supernatant fractions, obtained from either untreated or differentiated cells, KHT was produced when HHT was used as substrate. The addition of exogenous NAD, but not NADP, to incubations caused a significant increase in the production of KHT coincident with a decrease in the level of HHT. These data suggest that, in both differentiated and undifferentiated HL-60 cells, an NAD-dependent enzyme, apparently 15-prostaglandin dehydrogenase (15-PGDH), is expressed and catalyzes the conversion of HHT to KHT. In differentiated HL-60 cells, this metabolite is produced from arachidonic acid through a multi-enzymatic process involving the activities of cyclooxygenase, thromboxane synthetase and 15-PGDH. The production of KHT from arachidonic acid in undifferentiated HL-60 cells is probably limited, therefore, by the virtual absence of cyclooxygenase activity in these cells.