The present study examined differentiation-inducing activity by various tumor-necrosis-factor(TNF) mutants against the human leukemic cell lines HL-60 and U-937. Mutant TNF 471, from which 7 N-terminal amino acids of native TNF were deleted and Pro8, Ser9 and Asp10 were replaced by Arg, Lys and Arg, possessed the highest activity among the TNF mutants, and its activity was 120-fold that of native TNF. The various biological activities of TNF were signaled through 2 distinct receptors, p55 and p75. Although cytotoxicity was reported to involve mainly p55, this differentiation-inducing activity was not well understood. The fact that the affinity of TNF 471 was higher to p55 and lower to p75 than that of native TNF by a binding competition assay suggested that the differentiation-inducing activity was also signaled through p55. To verify this hypothesis, the human myelogenous leukemic cell line, KG-1, which scarcely expresses either receptor and does not differentiate with TNF, was transduced with the p55 or p75 gene. Subsequently p55 transfectants manifested a greater ability to differentiate; however, p75 transfectants did not differ from parental cells or from mock-transfectants. Further, the differentiation of p55 transfectants induced by TNF was reduced by the inhibitor of protein-kinase-C (PKC), staurosporine. These results indicate that the differentiation-inducing activity was signaled through the TNF receptor, p55, via PKC and that the excellent ability of TNF 471 to induce differentiation was related to its high affinity for p55.