B-cell human lymphoblastoid cell line, BALL-1, produced interferon-alpha (IFN-alpha) and cytotoxic factors by the stimulation of hemagglutinating virus of Japan (HVJ). These cytotoxic factors were identified to be tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta) on the basis of molecular level analysis. In the kinetic study, it was found that the maximum production of IFN-alpha was reached at 16-20 hr and that of TNF-alpha was at 10-16 hr after the stimulation by HVJ, whereas the maximum production of TNF-beta was at 40-50 hr, suggesting that the regulation of TNF-alpha and TNF-beta genes are different. These cytokines were purified by monoclonal antibody linked column chromatography, respectively. IFN-alpha was further separated into 9 subspecies on the basis of the difference of isoelectric point by FPLC chromatofocusing method. From the N-terminal amino acid sequence analyses of the 7 species of 9 subspecies, these subspecies were classified into 3 subtypes according to the Weissmann's notation. On the other hand, the amino acid sequence of the tryptic peptides of purified TNF-alpha and TNF-beta coincided well with the parts of the complete amino acid sequences which had been deduced from complementary DNA of TNF-alpha and TNF-beta of BALL-1 cell.