Effect of pentobarbitone on protein phosphorylation in rat brain in vitro was investigated. It was observed that at short term incubations pentobarbitone greatly enhanced incorporation of 32P into cerebral phosphoproteins which came to lower level after prolonged incubation. Dose response curve of the drug on protein phosphorylation was unconventional. The drug did not appreciably affect lipid peroxidation in brain homogenates. Effect of pentobarbitone on protein phosphorylation is not due to its inhibition of mitochondrial electron flow which would have given opposite results.