The ability of two subtypes of recombinant human alpha interferons (IFN), rIFN-alpha A and rIFN-alpha D, and two intramolecular hybrids, rIFN-alpha A/D and rIFN-alpha D/A, to modulate murine natural killer (NK) cell activity in vitro and in vivo was examined. The cytotoxic activity of murine spleen cells was markedly augmented by rIFN-alpha A/D following in vitro incubation, while the other IFN had little or no effect. The augmentation observed was dose dependent and inhibited by monoclonal antibody to rIFN-alpha A/D. Mice treated by one of several routes with rIFN-alpha A/D had elevated levels of NK activity in their spleen, peritoneal cavity, and peripheral blood following one to three daily injections. Augmentation of cytotoxicity was dose dependent in vivo and was less efficient or absent following treatment with the other recombinant IFN. When treatment was extended to ten or 12 daily i.p. injections, marked differences in NK levels resulted, depending upon the location from which cells were obtained. Following prolonged administration of rIFN-alpha A/D, a significant decrease in NK activity was seen with peripheral blood lymphocytes, while peritoneal cells retained elevated levels of activity; in spleen, NK activity was less than in mice treated for 3 days with rIFN-alpha A/D but greater than in control mice. Treatment of mice in vivo with IFN can either increase or decrease NK levels dependent upon both the length of treatment and the site at which the NK activity is measured. The use of rIFN-alpha A/D with murine cells is an excellent model to study the regulation of NK activity by IFN.