Natural killer cell-stimulatory factor or interleukin-12 (NKSF/IL-12) was originally identified and purified from the conditioned medium of Epstein-Barr virus (EBV)-transformed B-cell lines. Phorbol diesters were observed to be potent stimulators of NKSF/IL-12 production from the B-cell lines. Although monocytes were found to be the major producers of NKSF/IL-12 in peripheral blood (PB) in response to lipopolysaccharide (LPS) or to Staphylococcus aureus, several myeloid leukemia cell lines tested did not produce detectable NKSF/IL-12 either constitutively or upon stimulation with phorbol diesters. However, three lines, ML-3, HL-60, and THP-1, responded to LPS with significant levels of NKSF/IL-12 production, whereas S aureus was effective only on THP-1 cells. When the cell lines were preincubated with compounds known to induce them to differentiate, production of tumor necrosis factor alpha (TNF alpha) and IL-1 beta was in most cases maximal in cells with differentiated characteristics, whereas NKSF/IL-12 production in response to LPS in all three producing cell lines was significantly enhanced only by pretreatment with dimethylsulfoxide (DMSO) for 24 hours, or by costimulation with interferon gamma (IFN gamma). The efficiency of DMSO enhancement of NKSF/IL-12 production decreased after 2 to 5 days of incubation, when the cells acquired differentiated characteristics. Unlike DMSO, IFN gamma enhanced NKSF/IL-12 production, and IL-10 and dexamethasone inhibited it in cell lines and PB mononuclear cells stimulated by either LPS or S aureus. The ability of the cell lines to respond to these mediators of possibly physiologically relevant function provides a tissue-culture model for studying their mechanism of action.