The liver is a primary target for both acute and chronic effects of ethanol. Because ethanol is known to alter the function of guanine nucleotide regulatory proteins (G-proteins), changes in hepatic G-proteins could contribute to the adverse effects of ethanol on liver function. Male Wistar rats were fed a liquid diet containing 36% of calories as ethanol for 4 weeks. Control rats were pair-fed or allowed free access to a diet that isocalorically substituted maltose dextrins for ethanol. Liver plasma membranes were isolated and separated into basolateral and canalicular fractions by sucrose-density gradients. Enrichment of marker enzymes (5′-nucleoti-dase for canalicular membranes and forskolin-stimulated adenylyl cyclase activity for basolateral membranes) was not affected by ethanol feeding. Quantity of Gαs, and Gαi proteins in membrane fractions was determined by immunoblot. After ethanol feeding, immunoreactive Gαs protein was increased in liver plasma membranes compared with pair-fed controls. Gαi and Gαs were present in both the basolateral and canalicular fractions of the plasma membrane in control and ethanol-fed rats. Gαs quantity in the basolateral membrane was greater in ethanol-fed rats compared with controls, with no differences in Gαs observed in canalicular membranes. The quantity of Gαi did not change in response to ethanol feeding in any of the membrane fractions. Treatment of isolated plasma and basolateral membranes with 10 μmol/L 5′-guanylimidophosphate, a non-hydrolyzable guanosine triphosphate analogue that activates G-proteins, increased cAMP production to a greater extent in ethanol-fed rats compared with controls. These data indicate that ethanol increases the quantity and function of Gαs protein in rat liver plasma membranes. (HEPATOLOGY 1995; 21:1154–1160.)