The effects of ethanol on muscarinic receptor-mediated calcium responses were investigated in individual primary rat astrocytes and human 132 1N1 astrocytoma cells using indo-1/AM and image cytometry. After a 30-min incubation, carbachol-induced calcium responses were inhibited only at 100 or 250 mM ethanol. The effects of ethanol were more pronounced and occurred at lower concentrations with longer exposures, with significant inhibition seen at 10 mM following a 24-hr incubation. Thapsigargin- and glutamate-induced responses were unaffected by ethanol, indicating some selectivity in this inhibition. Upon removal of ethanol, inhibition of calcium responses persisted for up to 6-12 hr, with carbachol responses returning to control levels by 24 hr after washout. Ethanol exposure did not affect muscarinic-receptor binding in astrocytoma cells, but inhibited carbachol-induced IP(3) formation. Inhibition of (3)H-thymidine incorporation by ethanol also persisted upon removal of the alcohol, with a time-dependency similar to that of the calcium responses. These results indicate that ethanol inhibits muscarinic receptor-induced calcium responses in astroglia in a concentration- and duration-dependent manner. They also show that co-incubation with ethanol is not necessary for this effect, suggesting that long-term exposure to ethanol may modify, in a reversible manner, the coupling of muscarinic receptors with its effector. This effect of ethanol may play a role in ethanol's inhibition of carbachol-induced thymidine incorporation.