Abstract— The intracerebral injection of 32Pi into guinea‐pig cortex resulted in a steady rate of incorporation into all phospholipids over a 20 h period. The specific radioactivities of phosphatidate and phos‐phatidylinositol in synaptosomes prepared from cortex prelabelled, in vivo, were at a maximum after 2 h and the respective activities were 3–8 times higher than in whole cortex. This peak in labelling corresponded with the maximum specific activity of the brain ATP. No similar differential labelling pattern was observed for phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine. Electrical stimulation of the prelabelled synaptosomes produced a rapid drop in the specific activity of phosphatidylinositol and phosphatidate and an increase in the specific activity of CDP‐diacylglycerol. The specific activity of synaptosomal ATP was not affected. Study of the subsynaptosomal fractions obtained after osmotic rupture of the synaptosomes revealed that the most highly labelled phosphatidylinositol was in the synaptic vesicle fraction (D) and the most active phosphatidate was in a ‘microsomal’ fraction (E). Electrical stimulation caused a loss of phosphatidylinositol radioactivity from fraction D and a loss of phosphatidate radioactivity from fraction E. The specific activity of these lipids in other fractions was not affected. A possible role for presynaptic phosphatidylinositol is suggested.