Using a procedure in which poly-phosphoinositides (poly-PI) in C57B1 mouse brain were labeled with [32P]Pi or [32P]ATP, the effects of chronic ethanol administration and age on metabolism of these anionic phospholipids were examined. Within 4 h after intracerebral injection, both labeled precursors were effectively incorporated into membrane phospholipids with high proportions of labeling among phosphatidylcholine, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. With few exceptions, the phospholipid labeling patterns in different brain regions, e.g. cortex, hippocampus and hypothalamus, were similar. However, when the brain homogenate was subjected to differential and sucrose-Ficoll gradient centrifugation, different phospholipid labeling patterns were observed in the subcellular membrane fractions. Young adult mice given an ethanol (5% w/v) liquid diet for 2 months showed an increase in the levels of labeled phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate and phosphatidylserine in the cortex and hippocampus as compared to the pair-fed controls, but these changes were not observed in the hypothalamus. In another study, 12- and 26-month-old mice were administered either an ethanol (8 g/kg in two doses daily) or a control diet by gavage for 3 weeks. The 12-month-old group given the ethanol diet showed an increase in labeled poly-PI which was found largely in the synaptosomal fraction. Surprisingly, the 26-month-old mice given the same ethanol paradigm showed a decrease in labeled poly-PI. Consistent with our previous observations, the 26-month-old mice showed a higher proportion of labeled poly-PI in the synaptosomal fraction as compared to the younger age group. Taken together, these results suggest that chronic ethanol administration could result in an increase in biosynthesis of poly-PI in adult mice but aged mice showed a different response to the effect of ethanol.