The effects of nicotine and other cholinergic drugs on the initial incorporation of 32Pi, and [3H]glycerol into 1-phosphatidylinositol-4-phosphate(DPI)and 1-phosphatidylinsitol-3,4-bisphosphate (TPI) in the rat brain microsomal fraction were studied. Nicotine and eserine significantly decreased and mecamylamine increased [32P]- and [3H]TPI levels. Atropine had no effect on labeled TPI levels. Dose-effect studies for nicotine revealed that as little as 0.1 mg/kg of nicotine, i.p., significantly decreased the accumulation of labeled TPI. It was concluded that pharmacologically relevant and nonconvulsive doses of nicotine can markedly affect brain polyphosphoinositide synthesis and/or metabolism; the nature of the effects is consistent with theories suggesting that the conversion of TPI to DPI will release membrane lipid bound Ca2+ and, thus, increase membrane excitability.