To characterize the mechanism(s) by which 1,25-dihydroxyvitamin D3 (calcitriol) modulates the costimulatory capacity of monocytes, we examined the effect of calcitriol pretreatment of monocytes on their capacity to promote T cell proliferation (accessory cell function). Correlation of calcitriol-dependent changes in monocyte accessory cell function and alterations in phenotype and cytokine production, and the dependence of these changes on cell viability, were studied. Calcitriol pretreatment induced a defect in accessory cell function that was evident with fixed monocytes, suggesting a cell-surface—associated mechanism. Altered accessory cell function did not correlate with changes in HLA—DR antigen expression and was unaffected by concurrent treatment with interferon-γ. Calcitriol treatment did not alter either the expression of adhesion molecules or monocytic production of interleukin-1β (IL-1β) or IL-6. Exogenous IL-1 or IL-6 did not overcome the impaired costimulatory activity of calcitriol-treated monocytes. Thus, calcitriol treatment reduces the capacity of monocytes to promote lectin-induced T cell activation at the level of the plasma membrane, perhaps through altered expression of an uncharacterized molecule important in monocyte—T cell interactions. At chronically inflamed sites, elaboration of calcitriol by activated macrophages may regulate the ability of monocytes to induce both antigen-dependent and antigen-independent T cell proliferation.