1,25-Dihydroxyvitamin D3 (1,25(OH)2-D) has been shown to be a macrophage-derived cytokine, capable of regulating myeloid differentiation and T-cell activation in vitro. Therefore, we examined the effects of 1,25(OH)2-D on the monocyte phenotype and function of human peripheral blood monocytes as an index of its biologic role at an inflammatory site. 1,25(OH)2-D treatment consistently and specifically reduced HLA-DR and CD4 expression by monocytes, while CD14 and class I HLA antigen expression were unaffected. Expression of Fc gamma R I-III on monocytes was variably modulated by 1,25(OH)2-D treatment, but no differences in antibody-dependent cell cytotoxicity (ADCC) were observed, measured using either ADCC or anti-Fc gamma R-antibody expressing hybridomas. In contrast, the ability of monocytes to induce antigen-dependent T-cell proliferation was markedly reduced by 1,25(OH)2-D pretreatment for as little as 6 hours. Addition of interleukin-1 (IL-1), IL-6, or indomethacin did not restore antigen-dependent T-cell proliferation, suggesting that this observation was not secondary to changes in IL-1, IL-6, or PGE2 production induced by 1,25(OH)2-D. These data suggest that 1,25(OH)2-D treatment specifically modulates human monocyte phenotype and function, altering HLA-DR antigen expression and antigen presentation, while leaving lytic function intact. These findings may be relevant to the immunobiologic role of 1,25(OH)2-D.