Ethanol exposure alters signal transduction through the adenylyl cyclase (AC) system. To elucidate the basis for this effect, we investigated the AC system in peripheral lymphocytes from abstinent alcoholic men (n = 22), actively drinking alcoholic men (n = 41), and nonalcoholic control men (n = 16). Immunoblot analysis of lymphocyte membranes from abstinent alcoholics demonstrated a 3.0-fold increase in the level of Gi2 alpha protein (p < 0.05) compared with controls. However, levels of Gs alpha protein were similar in both groups. Abstinent alcoholics had a 2.9-fold increase in Gi2 alpha mRNA (p < 0.001) and a 2.7-fold increase in Gs alpha mRNA (p < 0.03) compared with lymphocytes from control subjects. Actively drinking alcoholics, in contrast, had unaltered Gs alpha protein, Gi2 alpha protein, and Gi2 alpha mRNA levels compared with control subjects, but did have a 1.8-fold increase (p < 0.01) in Gs alpha mRNA. Consistent with enhanced Gi2 alpha expression, lymphocyte membranes from abstinent alcoholics had decreased basal, prostaglandin E1-, guanosine 5'-0-(3-thiotriphosphate)-gamma S-, and forskolin-stimulated AC activity compared with both controls and actively drinking alcoholics (p < 0.05). We conclude that lymphocyte AC is reduced during abstinence from alcohol and enhanced expression of the inhibitory G-protein, Gi2 alpha, may account for this change.