While there have been reports on the in vitro effects of ethanol on mitogen-stimulated lymphocyte proliferation, little attention has been paid to whether these effects are mediated by the ethanol molecule per se or the associated hyperosmolality. There is controversy whether ethanol at high concentrations inhibits or has no effect on mitogen-stimulated proliferation of T cells. In this study we sought to distinguish between effects due to ethanol and those related to increased osmolality. Rat splenocytes were cultured in vitro in hypertonic (320 mOsm) or isotonic (320 mOsm) ethanol solutions (up to 400 mg/dL), or in corresponding hypertonic solutions without ethanol. Proliferative responses to the B cell mitogen lipopolysaccharide and the T cell mitogen concanavalin A were measured by mitochondrial dimethylthiazole diphenyl tetrazolium metabolism and [(3) H]-thymidine incorporation into DNA. B cell response was not significantly affected by ethanol under any conditions. By contrast, T cell proliferative responses were significantly inhibited by isotonic ethanol solutions or hypertonic solutions in the absence of ethanol, but not by hypertonic ethanol solutions. These results indicate that, while high osmolality is normally inhibitory to lymphocyte function, the increase in osmolality caused by ethanol is protective against the otherwise detrimental effects of ethanol.