Human polymorphonuclear leukocytes (PMN) stimulated by lipopolysaccharide (LPS) produce interleukin-12 (IL-12). Both the free IL-12 p40 chain and minute amounts of the biologically active IL-12 p70 heterodimers are produced by PMN. Interferon-gamma (IFN-gamma) enhanced the LPS-induced secretion of both the free IL-12 p40 chain and the p70 heterodimer by approximately fivefold. As observed for other IL-12-producing cell types, the ratio of free p40 chain to p70 heterodimer secreted by LPS-stimulated PMN was approximately 20:1. LPS induced a 100-fold increase of IL-12 p40 mRNA, but had minimal effect on p35 mRNA accumulation. IFN-gamma enhanced the LPS-induced accumulation of p40 mRNA and directly induced a several-fold increase in the accumulation of p35 mRNA. Therefore, the combined effect of LPS and IFN-gamma induced sufficient expression of both p40 and p35 to attain production of the biologically active p70 heterodimer at physiologically relevant concentrations. The ratio between p40 and p35 mRNA abundance in PMN stimulated with both LPS and IFN-gamma was approximately 200:1, explaining the secretion of the free p40 chain in much higher concentrations than the p70 heterodimer. IL-10, an inhibitor of the production of various cytokines in PMN, also suppressed IL-12 mRNA accumulation and secretion by PMN. Because of the important immunoregulatory function of IL-12, in particular induction of IFN-gamma production and facilitation of T helper cell type 1 response, the ability of PMN to produce IL-12 suggests that neutrophils may play an active role in the regulatory interaction between innate resistance and adaptive immunity.