The fusion of thioglycollate-elicited peritoneal macrophages from lipopolysaccharide (LPS) non-responsive C3H/HeJ mice to an HPRT-negative variant of the murine macrophage cell line P388D1 has resulted in the derivation of a series of macrophage hybrids. Following exposure to LPS, these hybrids now produce the cytokine hepatocyte-stimulating factor (HSF) which induces the synthesis of the acute-phase reactant alpha 2-macroglobulin in primary rat hepatocyte cultures. The concentration of extracellular HSF was dependent upon both the duration and amount of LPS, with optimal HSF being detected after 72 hr incubation with 10 micrograms/ml of LPS. Parallel LPS-stimulated cultures treated with 10(-6)M dexamethasone did not secrete detectable amounts of HSF. Both the molecular weight (29,000 MW), and the fact that HSF activity was not inhibited by an antiserum directed against murine interleukin-1 alpha (IL-1 alpha), suggests that HSF and IL-1 are distinct cytokines. Therefore, macrophage hybrids have been derived which have acquired the LPS-responsive phenotype and which synthesize the cytokine HSF following LPS stimulation. This phenotype appears stable since similar results have been observed with these hybrids after in vitro culture for over 8 months.