A human myelomonocytic cell line, HBL-38 cells, propagated in vivo, spontaneously produced interferon (IFN)-gamma and IFN-alpha. Whereas hemmagglutinating virus of Japan (HVJ) enhanced the production of IFN-alpha, bacterial lipopolysaccharide (LPS) markedly enhanced the production of IFN-gamma. LPS could be replaced with lipid A. Furthermore, the enhancement of production of IFN-gamma by LPS was completely abolished by polymixin B. IFN-gamma derived from LPS-stimulated HBL-38 cells was purified to homogeneity and characterized. The apparent molecular weight, subspecies composition, amino acid sequence and glycosylated sites were in agreement with those of the product of normal human peripheral blood lymphocytes (PBL). These results indicate that the myelomonocytic HBL-38 cells, not a T-cell line, can also produce IFN-gamma identical to the product of normal human PBL.