Three major subtypes of human interferon-alpha (IFN-alpha), isolated from virus-induced leukocytes, were compared for their antiviral and anticellular activities on one hand, and for their ability to induce (2'-5') oligoadenylate synthetase on the other hand. One subtype, IFN-alpha 1, was found to have low specific antiviral (6.10(6)-5.10(7) units/mg) and anticellular activities when measured on a variety of human cells. A second subtype, exhibiting an unusually high molecular weight (26,000) by SDS-polyacrylamide gel electrophoresis (IFN-alpha 26K), was found to have the highest known specific antiviral (8.10(8)-2.10(9) units/mg) and anticellular activities. Thus, these two subtypes of IFN-alpha differ by a factor of 330 and represent the two extremes in the antiviral scale on human cells. A third subtype, IFN-alpha 2, was tested as well and was found to have intermediate antiviral and anticellular activities. The ability of these three subtypes to induce (2'-5') oligoadenylate synthetase in human cells was then measured. It was found that on a weight basis, the three subtypes were equally effective in inducing the enzyme. Since the level of (2'-5') adenylate oligomers is affected also by the interferon-induced (2'-5') phosphodiesterase, the ability of these subtypes to induce this enzyme was compared as well and was found to be very similar. We therefore conclude that the differences in potency between these IFN-alpha subtypes are not related to their ability to induce (2'-5') oligoadenylate synthetase.