Abstract: Phosphatidylethanol is formed by phospholipase D in animal cells exposed to ethanol. Previous reports have demonstrated that the degradation of phosphatidylethanol is slow, indicating that this lipid may be present in the cells after ethanol itself has disappeared. Accumulation of an abnormal alcohol metabolite may influence cellular functions. In the present study, cultivation of NG108–15 neuroblastoma × glioma hybrid cells in the presence of ethanol resulted in an accumulation of phosphatidylethanol and a simultaneous increase in basal inositol 1,4,5‐trisphosphate levels. The direct effects of phosphatidylethanol on the phosphoinositide signal transduction system were examined through incorporation of exogenous phosphatidylethanol into membranes of ethanol‐naive cells. An incorporation amounting to 2.8% of cellular phospholipids was achieved after a 5‐h incubation with 30 μM phosphatidylethanol. Phosphatidylethanol was found to cause a time‐and dose‐dependent increase in the basal levels of inositol 1,4,5‐trisphosphate. The effects on inositol 1,4,5‐trisphosphate levels of exogenously added phosphatidylethanol and ethanol exposure for 2 days were not additive. No effect on bradykinin‐stimulated inositol 1,4,5‐trisphosphate production could be detected. However, the increase in basal inositol 1,4,5‐trisphosphate levels indicates that phosphatidylethanol affects inositol 1,4,5‐trisphosphate turnover and emphasizes the importance of considering phosphatidylethanol as a possible mediator of ethanol‐induced effects on cellular processes.