1. The in vitro incorporation of 35S-methionine into actin and total soluble proteins, as well as the levels of actin mRNA, were studied in the hypothalamus and frontal cerebral cortex of adult male rats killed at six different time intervals during a 24-hr cycle. 2. The specific activity of total soluble proteins after labeled methionine incubations did not vary as a function of time of day in any of the examined brain regions. 3. The incorporation of 35S-methionine into a 43-kDa protein, corresponding to the electrophoretic mobility of actin, varied diurnally in the hypothalamus, exhibiting a maximum at 1200 hr. Such a diurnal variation was not found in frontal cerebral cortex. 4. Similar results were obtained when labeled methionine incorporation into actin was assessed in hypothalamus and cerebral cortex by an immunoprecipitation procedure. 5. An increase in actin hypothalamic mRNA levels, quantitated by dot-blot analysis, was found at 0800, 4 hr in advance to the maximum in 35S-methionine incorporation to actin. 6. The levels of actin mRNA did not vary significantly as a function of time of day in the frontal cerebral cortex.