To elucidate the differentiation-associated expression of enzymes catalyzing arachidonic acid metabolism, we measured arachidonate metabolites by reverse-phase high pressure liquid chromatography in monocytoid leukemia (ML-1, THP-1, and U937) and myeloid leukemia (KG-1) cell lines. Undifferentiated ML-1 or THP-1 cells produced trace amounts of eicosanoids via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. Upon differentiation induced by phorbol ester (phorbol 12- myristate 13-acetate [PMA]), metabolites via the COX pathway were increased by 100-fold in ML-1 and THP-1 cells, while the LOX products remained barely detectable. All the COX metabolites were elevated, but thromboxane A2 (TXA2) formation was threefold higher in ML-1 cells than in THP-1 cells. Similar time-related increases in COX metabolites were observed in THP-1 cells induced to differentiate with retinoic acid. Undifferentiated U937 cells were capable of generating a much higher quantity of COX products than ML-1 or THP-1 cells, but, upon PMA- induced differentiation, COX products were increased by only two-fold to threefold over the undifferentiated cells and the total COX products in differentiated U937 cells were only one-seventh of those produced by differentiated ML-1 or THP-1 cells. KG-1 cells had an entirely different metabolic profile. They produced a large quantity of a metabolite coeluted with prostaglandin D2, and PMA had no effect on inducing changes in arachidonic acid (AA) metabolism. Increased COX metabolite formation in differentiated THP-1 and ML-1 cells was due to an enhanced level of prostaglandin H synthase enzyme mass, as measured by Western blot analysis. The TXA synthase activity was also increased by approximately 100-fold in PMA-induced ML-1 cells and 10-fold in THP- 1 cells. These findings indicate that increased expression of prostaglandin H and TXA synthase enzymes is a feature of differentiated monocytoid leukemia cell lines.