Nine interferon-alpha subtypes, IFN-alpha1, IFN-alpha2, IFN-alpha5, IFN-alpha7, IFN-alpha8, IFN-alpha10, IFN-alpha14, IFN-alpha17, and IFN-alpha21, were separated from purified human lymphoblastoid IFN. We tested their inhibitory effects on cell growth and replication of Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV) and their induction of 2',5'-oligoadenylate synthetase (2', 5'-OAS) in ACHN renal cell carcinoma cells. In terms of all three activities, the nine subtypes had similar relative activities, with IFN-alpha10 the most active and IFN-alpha1 the least. Their relative effects on cell growth were similar in two other human cell lines, SK-LU-1 lung cancer cells and KU-2 renal cell carcinoma cells, whereas cells of the Daudi Burkitt lymphoma line behaved quite differently, being highly sensitive to all the nine subtypes. The relative effects with ACHN cells correlated well with their relative binding affinities. However, each of the subtypes bound to both ACHN and Daudi cells to almost the same extent. This suggests that their profound inhibitory effects on the growth of Daudi cells are amplified at some stage in the signal transduction pathway or in the expression of genes that results from binding to the IFN-alpha receptor.