1 alpha,25 dihydroxyvitamin D3 (1,25 D3) is known to interact with hematopoietic cells. The purpose of this study was to determine the effect of 1,25 D3 on hydrogen peroxide (H2O2) production and tumor cell killing by mouse peritoneal macrophages and human blood monocytes. Enhanced monocyte and macrophages phorbol myristate acetate (PMA)-stimulated H2O2 production was observed at concentrations of 0.13 to 130 nM 1,25 D3 and and was maximal at 1.3 nM. At concentrations of 100 U/ml, gamma interferon (IFN-gamma) alone had a similar effect but, in combination with 1,25 D3, there was no cooperative effect. At concentrations ranging from 0.13 to 130 nM, 1,25 D3 failed to augment tumor cell lysis by macrophages from peptone-injected normal or bacillus Calmette-Guerin (BCG)-infected mice, or by blood monocytes from normal humans. Our results indicate that 1,25 D3 can activate the monocyte and macrophage for H2O2 secretion without concomitant activation for tumor cell killing.