Previous studies in this laboratory indicate in vivo administration of a single dose of ethanol (EtOH) can decrease basal and polyinosinic/polycytidilic acid-stimulated natural killer (NK) cell activity. In the present study, in vivo administration of EtOH also decreased interleukin-2-induced splenic NK cell activity measured 12 hr after administration of EtOH and interleukin-2. In vitro experiments were conducted in which the concentration and duration of exposure to interleukin-2, EtOH and the major metabolites of EtOH (acetaldehyde and acetate) were designed to match the conditions measured or expected in the in vivo experiments. EtOH and acetaldehyde decreased NK cell activity in a dose-dependent manner, but concentrations equivalent to those reported in blood caused relatively small decreases (< 20%) in NK cell activity. A combination of EtOH, acetaldehyde and acetate also suppressed NK cell activity, but suppression by concentrations comparable to those expected in vivo was less than 20%. By comparison, exposure to EtOH at similar concentrations and for similar times in vivo causes approximately 50% suppression of NK cell activity. Thus direct action of EtOH and its metabolites is not the primary mechanism by which NK cell activity is suppressed after administration of EtOH in vivo. This result suggests that other mechanisms, such as the induction of immunosuppressive neuroendocrine mediators, should be investigated.