In order to observe the effects of morphine on protein metabolism in mouse brain, experimental procedures were carried out over a 7 hr period of infusion. When the analgesia reached a peak, namely around 2 hr after the start of infusion, the in vivo incorporation of radioactive leucine into protein estimated by the dual label technique was uniformally depressed in all the examined subcellular fractions of both brain and liver. After tolerance developed, however, the incorporation of leucine increased to a much higher level than the control in brain subcellular fractions and the increase was masked by naloxone. In contrast, the incorporation into a TCA soluble fraction of the brain S2, separated by Gray and Whittaker's method, was more than doubled even after 4 hr infusion. While the in vitro incorporation rate of the mitochondrial fraction significantly fluctuated during development of tolerance with naloxone that of the synaptosomal fraction did not fluctuate. The observed coincidence in the time course of development of tolerance and changes in the brain protein synthesis indicates a possible relationship between the phenomena, though the causal nature of the relationship could not be elucidated.