The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3), has been shown in both in vitro and in vivo experiments to be immunoregulatory. We analyzed the effects of the hormone on the human mixed lymphocyte reaction (MLR), the in vitro model of allograft response. Suppressor-cell activity of MLR-generated effector cells was enhanced by calcitriol (10(-10) to 10(-8) M). This suppressor activity was nonspecific since calcitriol-generated effector cells could suppress a primary MLR with stimulators and/or responders heterologous to the effector-generating MLR. Calcitriol (10(-9) to 10(-7) M) was also effective in preventing the generation of cytotoxic T cells when tested in a 51Cr release assay. While no differences were observed in the phenotypic analyses of the MLR-generated effector cells between 1,25-D3-treated cells and control cells, a significant reduction of class II antigen expression was observed in the presence of the hormone. The effects of calcitriol on human MLR are similar to those observed with cyclosporine.