Both interferon-gamma (IFN-gamma) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) induce changes in the human monocytic cell line U937 that may reflect cellular differentiation. The effects of recombinant IFN-gamma and 1 alpha,25(OH)2D3 on U937 cells with regard to the release of superoxide anion (O2-), prostaglandin E2 (PGE2), and mononuclear cell factor (MCF) after stimulation with phorbol myristate acetate (PMA) were examined. PMA did not induce O2- production in untreated cells. A 3-day preincubation with IFN-gamma or 1 alpha,25(OH)2D3 resulted in a 5- to 10-fold increase in PMA-stimulated production of O2- as compared to cells preincubated in medium alone. The response was related to IFN-gamma and 1 alpha,25(OH)2D3 concentrations. In contrast, the PMA-induced production of PGE2 and MCF does not require preincubation with either IFN-gamma or 1 alpha,25(OH)2D3. These results suggest that O2- production and cytokine production (i.e., PGE2 and MCF) are modulated by different signals related to maturation processes.