Recent evidence suggests that adenosine mediates many of the acute and chronic effects of ethanol in both cultured cells and whole animals. These adenosine-mediated effects of ethanol result from ethanol-induced increases in extracellular adenosine. Acute exposure of primary cultures of rat hepatocytes to 12.5-200 mM ethanol increased extracellular adenosine concentrations by 20-35%. Pretreatment of hepatocytes with 100 microM 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, completely blocked ethanol-induced increases in extracellular adenosine at 12.5 and 25 mM ethanol. However, even in the presence of 4-methylpyrazole, ethanol at concentrations greater than 50 mM still increased extracellular adenosine concentrations. This increase appears to be due to ethanol inhibition of adenosine uptake via the nucleoside transporter (50% inhibitory concentration, 28 mM). After chronic treatment with 100 mM ethanol for 48 h, acute challenge with ethanol no longer inhibited adenosine uptake, i.e., the nucleoside transporter had become tolerant to ethanol. Moreover, in these chronically treated cells, ethanol-induced increases in extracellular adenosine were completely blocked by treatment with 4-methylpyrazole at all concentrations of ethanol. Taken together, these results suggest that increased extracellular adenosine in hepatocytes is dependent on both ethanol oxidation and inhibition of adenosine uptake via the nucleoside transporter.