Cytosolic extracts prepared from perfused whole liver or purified hepatocytes of C57BL/6 mice inhibited interleukin-2-and concanavalin A-induced spleen cell proliferation in uitro. In contrast, cytosolic extracts from purified nonparenchymal liver cells had no effect. Arginase and very-low-density lipoprotein were previously identified as two immunoinhibitory substances present in liver cytosolic extracts. W e demonstrated, however, that inhibitory activity remained after removal of very-low-density lipoprotein and arginase from liver cytosolic extract by repeated ultracentrifugation and gel filtration chromatography, respectively, suggesting the presence of another inhibitor. Further purification by anion-exchange chromatography and chromatofocusing led to the isolation of a novel liverderived immunoinhibitory factor. This liver-derived immunoinhibitory factor is sensitive to pronase di- gestion and heat and acid treatment; it has an estimated isoelectric point of 8.25. The M, of liver-derived immunoinhibitory factor is 28 kD as estimated from its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is identical under both reducing and nonreducing conditions, indicating a monomeric nature of this protein. Amino acid composition analysis discloses that liver-derived immunoinhibitory factor is relatively rich in glycine and proline residues. Interleukin-2-induced spleen cell proliferation in uitro is inhibited by this liver-derived immunoinhibitory factor, with a 50% inhibitory dose of 1 . 4 nmoUL. Furthermore, the biological activity of the liver-derived immunoinhibitory factor is not confined to mouse spleen cells, since the growth of B16 mouse melanoma and H35 rat hepatoma cells is also inhibited. A comparison with other liver-derived immunoinhibitors reported previously supports our claim that the liver-derived immunoinhibitory factor is a novel inhibitory protein. (HEPATOLOGY 1991;14:888-894.)