Five human interferon-alpha (leukocyte) subtypes derived from genes cloned in Escherichia coli have been compared for their ability to induce antiviral activity against vesicular stomatitis virus infection of various mammalian cell cultures. These interferons, designated LeIF-A (IFN-alpha 2), -B, -C, -D (IFN-alpha 1) and LeIF-F, show different relative activities when assayed on human, bovine, hamster, mouse, rabbit and monkey cell lines. As with a natural human buffy-coat interferon-alpha preparation, three subtypes (LeIF-B, -C and -D) showed considerable activity on RK-13 rabbit cells, but two (LeIF-D and -F) also showed some activity on mouse L-929 cells. Of the five interferon subtypes examined, LeIF-F demonstrated the highest degree of species specificity.