HL-60, a promyelocytic cell line, was treated with both recombinant interferon-gamma (IFN-γ) and 1,25-(OH)2 vitamin D3 (D3), and the effect on monocyte-specific markers was assessed. IFN-γ and D3 modulated different stages in the monocytic differentiation with respect to interleukin-1 (IL-1) production and secretion. D3 induced production of an intracellular IL-1 activity that was not secreted after lipopolysaccharide stimulation. IFN-γ did not induce intracellular IL-1 but differentiated HL-60 cells, which had been treated with D3, so that lipopolysaccharide stimulated IL-1 release. Both D3 and IFN-γ individually enhanced expression of nonspecific esterase; in combination the two agents potentiated each other. Expression of cell surface Leu-M3 antigen was also enhanced by the combination of these two agents. Thus, IFN-γ not only potentiated expression of D3-induced markers but also conferred phenotypic properties characteristic of monocytes. IFN-γ may play a role in the differentiation of bone marrow cells to mature monocytes.