Nitric oxide (NO.), a free radical gas, has been implicated in the CNS actions of ethanol. The brain contains several cell types that can produce NO., including neurons and glia. This study examined the effect of acute and chronic ethanol exposure on the activity of the inducible isoform of nitric oxide synthase (iNOS) found in neuroglia. Experiments were performed using intact rat C6 glioma cells, and NO. production was assessed by nitrite accumulation after iNOS induction by coadministration of phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Ethanol was inhibitory at high concentrations (IC50 approximately 150 mM) when acutely present during the 24-hr period subsequent to initiation of enzyme induction. In contrast, cells exposed to ethanol were inhibited chronically at clinically relevant lower concentrations (IC50 approximately 30 mM with 10 days exposure). Chronic inhibition was both time- and concentration-dependent. Inhibition by ethanol seems to be a consequence of interference with LPS signal transduction. Acutely, ethanol did not affect the ability of PMA to synergize with LPS to induce activity, but it attenuated the ability of LPS to synergize with the PMA. Ten days exposure to 50 mM ethanol decreased the LPS potency by 4-fold in the presence of a maximally activating concentration of PMA, although not significantly changing PMA potency. Inhibition by chronic ethanol exposure was long-lasting, being retained over 24 hr in cells returned to control conditions. Thus, chronic ethanol may downregulate key components needed for iNOS expression.(ABSTRACT TRUNCATED AT 250 WORDS)