The physiological agents gamma interferon and 1,25 dihydroxyvitamin D3 (calcitriol) are both potent inducers of monocytoid differentiation in the HL-60 promyelocytic leukemia cell line. However, the populations resulting from treatment with either inducer have differing properties, suggesting that these agents do not have identical modes of action. In this study we have compared the effects of gamma interferon (IFN gamma) and calcitriol and examined the effect of exposing HL-60 cells to both compounds simultaneously. In addition, we have examined the effects of inhibitors of protein and RNA synthesis on induction of cell surface antigens in order to gain insight into the mechanisms of action of these compounds. We found that combining IFN gamma and calcitriol resulted in greater monocytic differentiation of HL-60 cells than did either compound alone. In addition, a monocyte-associated cell surface antigen, My23, and class-I HLA antigen were induced by both agents in at least an additive manner. In contrast, combined treatment with both compounds did not augment antibody-dependent cellular cytotoxicity (ADCC) any better than did IFN gamma alone, but it did cause a decrease in the density of receptors for the Fc portion of immunoglobulin. In studies examining the effects of IFN gamma and calcitriol on the cloning potential of HL-60 cells, we found that incubation of cells with either compound alone significantly reduced the number of HL-60 colonies, and both compounds added together caused an almost total elimination of colony-forming cells. Inhibition of RNA synthesis with alpha amanitin had no effect on the action of IFN gamma on cell surface HLA, but did block the induction of My23 antigen, suggesting that the mechanism of HLA induction may involve posttranscriptional phenomena. RNA-synthesis blockade inhibited the ability of calcitriol to induce My23. These studies demonstrate that multiple and separable events accompany the monocytoid differentiation of HL-60 cells induced by different chemical mediators and that different intracellular pathways may be involved.