Abstract In vivo protein phosphorylation was examined in postsynaptic density-enriched fractions isolated from rat brain. In vivo phosphorylation was carried out by injecting rats intraventricularly with [ 32P]orthophosphate followed by isolation of postsynaptic densities from pooled cerebral cortices. In vivo 32P-labeled postsynaptic densities were then fractionated by sodium dodecylsulfate-polyacrylamide slab gel electrophoresis and stained with Coomassie Blue. The protein banding pattern was typical for postsynaptic densities. The principal polypeptide component occurred in a single band at an apparent molecular weight of 51,000. Autoradiographs of the dried gels showed a major peak of radioactivity associated with the 51,000 molecular weight component for the in vivo labeled postsynaptic density fraction. Additional minor peaks of radioactivity were also observed. These results represent the first demonstration that proteins associated with the postsynaptic density readily incorporate phosphate in vivo and may represent a major and important class of synaptic phosphoproteins.