The ability of nontoxic monophosphoryl lipid A (MPL) to stimulate nonspecific resistance against viral infection was investigated. Mice pretreated intravenously with squalane-in-water emulsions of MPL, alone or in combination with other immunostimulants, were given an aerosol of influenza virus three weeks after the pretreatment. Complete protection against lethal influenza virus infection was conferred when MPL was combined with trehalose dimycolate (TDM). The protective activity of MPL plus TDM combination was corroborated by a significant reduction of the lung virus titers. Combination of lower doses of MPL with TDM extracted from Mycobacterium bovis, but not with that of M. phlei, induced significant resistance to influenza virus. Preparations containing MPL alone, or combined with mycobacterial cell wall skeleton or muramyl dipeptide, were not effective. The adjuvant activity of MPL on bivalent influenza subunit vaccine was also studied. The primary antibody responses to influenza A and influenza B antigens were enhanced by the addition of MPL and were higher than the vaccine associated with aluminum hydroxide. The adjuvant activity of MPL was confirmed by the elevated secondary response. High levels of circulating antibodies were still present in the MPL group when antibody titers in the controls were waning.