The effect of long-term ethanol exposure on muscarinic receptors was investigated in human neuroblastoma SH-SY5Y cells. Exposure to 100 mM ethanol for 4 days enhanced both peak and steady-state levels of carbachol-stimulated inositol 1,4,5-bisphosphate increase. An ethanol concentration of 50 mM was sufficient for an enhancement of this event. The carbachol-stimulated decrease in [3H]inositol-labeled [3H]phosphatidylnositol 4,5-bisphosphate and increase [3H]inositol trisphosphate and [3H]inositol bisphosphate were also potentiated in ethanol-treated cells, which demonstrated that the receptor-stimulated activation of phospholipase C is augmented. Experiments with pirenzepine showed that carbachol-stimulated inositol 1,4,5-trisphosphate increase is mediated via M1 receptors both in ethanol-treated and control cells. Ethanol exposure for 2 or 4 days also caused an increase in [3H]N-methylscopolamine and [3H]quinuclidinyl benzilate binding sites and elevation of [3H]pirenzepine binding, which indicated that the number of muscarinic M1 receptors is increased in ethanol-treated SH-SY5Y cells. These results demonstrate that long-term exposure to ethanol potentiates muscarinic M1 receptor-stimulated activation of phospholipase C in SH-SY5Y cells. This is likely to be explained by an increased number of muscarinic M1 receptors.