Previous work has shown that freshly isolated lymphocytes from alcoholic subjects show significantly reduced basal and adenosine receptor-stimulated cAMP levels. This decrease could be due to ethanol-induced cellular adaptation or to a genetic difference in the regulation of cAMP signal transduction. Therefore, we cultured human lymphocytes in defined medium without ethanol for 7-8 days and then examined differences in receptor-dependent cAMP accumulation between lymphocytes from alcoholic and nonalcoholic subjects. After four to six generations in culture without ethanol, lymphocytes from alcoholic subjects have significantly higher cAMP levels than do cells from nonalcoholic subjects. Thus, a difference in cAMP signal transduction is demonstrable in cells from alcoholic subjects grown without ethanol. We also found that cultured lymphocytes from both alcoholic and nonalcoholic subjects show a decrease in receptor-stimulated cAMP levels after exposure to 200 mM ethanol for 48 hr. To determine whether alcoholic subjects have increased sensitivity to ethanol, lymphocytes were exposed to only 100 mM ethanol for 24 hr. Under these conditions, receptor-dependent cAMP levels did not change in cells from nonalcoholic subjects. However, lymphocytes from alcoholic subjects showed a 39% decrease (P less than 0.003) in adenosine receptor-stimulated cAMP levels. Taken together, the results show that (i) chronic ethanol treatment in culture reproduces the suppression of cAMP levels found in circulating lymphocytes from alcoholic subjects and (ii) despite four to six cell divisions in culture without ethanol, lymphocytes from alcoholic subjects exhibit significantly increased adenosine receptor-dependent cAMP levels and increased sensitivity to chronic exposure to ethanol. These findings suggest that the suppression of cAMP levels observed in freshly isolated lymphocytes from alcoholic subjects results from both a direct effect of chronic exposure to ethanol and a genetic difference leading to altered cAMP signal transduction.