In acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA) triggers cell differentiation, while arsenic trioxide (As2O3) generates partial differentiation and apoptosis. Animal and human studies suggest that newly diagnosed APL patients can be cured using As2O3 combined with ATRA. Cyclooxygenases are involved in prostaglandins and thromboxane synthesis. We have recently demonstrated that ATRA induces cyclooxygenase-1 (COX-1) expression and prostaglandin synthesis in NB4 cells and in blasts from patients with APL. In the present study we investigated the effect of ATRA and As2O3 co-treatment on COX-1 expression and prostaglandin formation and tested the effect of the COX-1/COX-2 nonselective inhibitor indomethacin on cell differentiation. Arsenic treatment of NB4 cells resulted in a partial but significant reduction of ATRA-dependent induction of COX-1 expression and activity. Pretreatment of NB4 cells with indomethacin significantly impaired ATRA/As2O3-induced differentiation, as assessed by cell morphology, nitroblue tetrazolium test or CD11c expression. PGE2 reversed the negative effect of indomethacin on differentiation of ATRA/As2O3-treated NB4 cells. In conclusion, COX-1 contributes to ATRA-dependent maturation of NB4 cells and is affected by As2O3. These results also suggest that nonsteroidal antiinflammatory drugs should be avoided in APL patients treated with the combination of ATRA and As2O3.