The effect of 17 beta-estradiol (E) on the oxidative metabolism of U937 cells was studied. E had no direct effect on the proliferation, surface adherence, or luminol-enhanced luminescence (LEL) of the U937 cells. Exposure of U937 cells to lymphocyte-conditioned medium (LCM) and 1,25-dihydroxyvitamin D3 allowed a maximal LEL response by cells stimulated with phorbol myristate acetate. In contrast, LCM from lymphocytes exposed to E (LCM-E) did not stimulate LEL to the same extent as did an equal volume of LCM. Increasing the E concentration in the lymphocyte medium was associated with a dose-dependent reduction in the LEL response of the U937 cells. Mixing equal quantities of LCM and LCM-E significantly decreased LEL levels. LEL stimulated by LCM, gamma-interferon, or differentiation-inducing factor was reduced by the presence of LCM-E. The inhibitory action of LCM-E was reversible and metabolite specific. 17 alpha-E produced an effect that was only one tenth the magnitude of the E effect. These findings indicate that E can modulate the differentiation of phagocytes indirectly by altering the synthesis and/or secretion of lymphokines.