Biological activities of human tumor necrosis factor-alpha (hTNF-alpha) and its mutants were compared. In cytotoxicity assay with L929 cells, one mutant, designated as TNF-B, showed 4.5-fold higher activity than TNF examined. And TNF-B also increased cytostatic activity and decreased protein synthesis against U937 cells. In receptor binding assay, TNF-B had almost the same affinity for TNF receports on L929 cells as hTNF-alpha. But another mutant, TNFarg, markedly decreased the all activities of hTNF-alpha and had lower affinity for receports on different types of target cell. These results indicated that the relative activity of TNFs to target cells may correlate with their affinity for receports. We also found that TNF-B retained the cytotoxicity of hTNF-alpha for HEp-2 cells. TNF-B also had two-fold higher affinity than hTNF-alpha for receptors on HEp-2 cells (only carrying hTNF-R55) and lower affinity for receptors on U937 cells (expressing mainly hTNF-R75). These results suggested that TNF-B might still interact with the human TNF-R55 receptor, but it might largely lose its ability to bind to human TNF-R75. Changes of biological activity of TNFs might be due to an altered affinity to the different types of TNF receptor on the target cells.