Ethanol (20% w/v) given to female, C57BL/6 mice in their drinking water was previously shown to suppress natural killer (NK) cell cytolytic activity in a mixed splenocyte population. The present study was designed to examine the hypothesis that ethanol consumption independently results in inhibition of NK cell cytolytic activity. Mice were given 20% w/v ethanol in the drinking water for 2 weeks, and splenic NK cells were enriched up to 88% based on surface expression of NK1.1. Cytolytic activity of these freshly enriched NK cells from ethanol-consuming mice against YAC-1 lymphoma cells was inhibited an average of 41% relative to water-drinking controls. Cytolytic activity of enriched NK cells from ethanol-consuming mice was stimulated to levels equal to control water-drinking mice after 16- to 18-hr incubation in 1000 units/ml recombinant interleukin 2. These data indicate that in vitro cytolytic activity of NK cells from ethanol-consuming mice is inhibited in the absence of other modulatory leukocytes.