Human natural killer (NK) cells exposed to the influenza surface antigen neuraminidase (NA) show high cytotoxic activity, as evaluated using chromium-labeled K562 target cells in a standard overnight cytotoxicity assay. The role of interferon (IFN) in the stimulation of NK cells was examined by using three separate approaches. The use of appropriate antibodies to check IFN- and NA-specific cell-mediated cytotoxicity (CMC) stimulation showed that antibodies to IFN (-alpha, -gamma) did not alter NA-induced CMC, and vice versa. The treatment of NK cells with actinomycin D, before or after stimulation with IFN and NA revealed that only IFN-induced CMC was inhibited (50 to 100%). However, NK cells that were stimulated with NA before their exposure to actinomycin D became susceptible to stimulation by IFN. The interaction kinetics between IFN and NA demonstrated the presence of two mechanisms of CMC stimulation. Taken together, the results clearly show that stimulation of CMC by a viral component is effected through an IFN-independent pathway, and that this mechanism is probably followed by IFN under certain conditions.