Previous studies have indicated that the pulmonary natural killer (NK) cells are important in early defense against influenza virus (PR8/34) infection by the intratracheal route. Since the natural route of virus entry into the lung is via the upper respiratory tract, the present study was to elucidate the role of NK cells in early defense to intranasal (IN) inoculation of influenza virus. Rabbit anti-Asialo GM1 (RAGM1) serum was administered by IN, intravenous (IV), or both routes, 24 hours before IN infection with a median lethal dose (LD50) of PR8/34 previously shown to kill 50% of the inoculated mice (B6D2F1 or nu/nu) by day 8. IN or IV inoculation of 20 microliters of RAGM1 (whole rabbit serum) had no effect on the survival of the mice to virus administered by the IN route. When RAGM1 was given IV (10 microliters) and IN (10 microliters) 24 hours before PR8/34 IN, 60% of the mice (B6D2F1 or nu/nu) died by day 4. Influenza virus titers were at least one log higher in the lungs of B6D2F1 mice and two logs higher in IN/IV NK-depleted nu/nu mice than in lungs of mice who received RAGM1 by IV or IN route alone or who were untreated. NK activity was depleted in lungs but not blood of mice treated IN with RAGM1 and was depleted in both lungs and blood if RAGM1 was given IV. These data support the hypothesis that NK cells are important to early defenses against influenza virus in the upper respiratory tract and that they are derived from both a local and systemic population of NK cells.