Human interferons from various sources have been characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by electrotransfer onto nitrocellulose and reaction with specific polyclonal and monoclonal antibodies. When gel slices were extracted, α-interferon subspecies possessed antiviral activity predominantly in the 18.6–19.7K region bands, the 3-interferon in the 22.1K band, and -γ-interferon in the 16.5–18.0K bands. Three of the monoclonal antibodies (Ab 138, Ab 126, Ab 098) reacted with a characteristic triplet of biologically active bands (18.6K. 19. 1K. 19.7K) obtained using the Namalwa cell interferons, while two (Ab 194 and Ab 232) reacted only with the 18.6K band and Ab 523 reacted with the 19.7K band. With the human leukocyte interferons, Ab 098. Ab 194. and Ab 232 reacted with the active 18.6K band. The Ab 138, Ab 126, and Ab 523 reacted specifically with certain lower molecular weight active bands (13K region). A comparison of the antiviral activity and reactivity towards monoclonal and polyclonal antibodies presents a differentiation of the subspecies of interferons in the wide array of closely related proteins in interferon preparations packaged for clinical use.