Glutamate is the primary excitatory neurotransmitter in brain. By stimulating neuronal activity, glutamate increases cellular energy utilization, enhances ATP hydrolysis and promotes the formation of adenosine. Adenosine has receptor-mediated effects that reduce or oppose the excitatory effects of glutamate. As a possible mechanism for ethanol's ability to inhibit excitatory effects of glutamate and enhance inhibitory effects of adenosine, we tested the hypothesis that ethanol promotes [3H]glutamate uptake and inhibits [3H]adenosine uptake. Using primary cultures of rat astrocytes, we found that acute treatment with ethanol (50 mM, 30 min) inhibited [3H]glutamate uptake and reduced protein kinase C (PKC)-induced stimulation of [3H]glutamate uptake. Prolonged treatment (50 mM, 3 day) with ethanol, however, increased both [3H]glutamate uptake and PKC activity. Contrary to other cell types, neither acute or chronic ethanol exposure affected [3H]adenosine uptake in astrocytes. These data indicate that in rat cortical astrocytes ethanol affects [3H]glutamate uptake but not [3H]adenosine uptake by affecting PKC modulation of transporter activity.