Previous studies have demonstrated that splenic natural killer (NK) cell activity of mice can be efficiently enhanced by polyadenylic.polyuridylic acid [poly(A).poly(U)]. Moreover, inoculation of mice with the duplex leads to the production of interferon (IFN). The NK boosting effect in mice was analyzed in parallel with the assay of an enzyme marker for the production and action of IFN, pppA(2'p5'A)n synthetase (2-5A synthetase). Kinetic studies revealed that splenic mononuclear cells of C3H/He mice inoculated intravenously with 10 micrograms or more of poly(A).poly(U) showed significantly enhanced cytotoxic activity in an in vitro 51Cr-release assay using NK sensitive YAC-1 target cells. Intravenous, intraperitoneal, and intramuscular administration of the duplex into mice were equally effective in obtaining such NK boosting effect. Furthermore, repeated weekly injections of poly(A).poly(U) did not induce resistance against NK boosting in the organisms. The levels of 2-5A synthetase in the organs, particularly spleens, of mice inoculated similarly with poly(A).poly(U) were greatly increased with a dose-dependent pattern. Repeated injections of mice with the duplex at 4-day intervals resulted also in significantly enhanced levels of splenic 2-5A synthetase after each injection. Thus, as a whole, enhanced NK activity accompanied increased levels of 2-5A synthetase in mice treated with poly(A).poly(U).