[(14)C]Phenylacetate was designed as a prototype tracer for the measurement of glial metabolism, and its potency in comparison with that of [(14)C]acetate was evaluated in this study. Normal rats were intravenously injected with [(14)C]phenylacetate or [(14)C]acetate, and radioactivity concentrations were measured in the plasma, cerebral cortex, cerebellum and peripheral tissues by dissection method. In addition, [(14)C]phenylacetate uptake in the rat brain was compared by autoradiography with that of [(14)C]acetate following the injection of fluorocitrate, a selective glial toxin, into the brain. [(14)C]Phenylacetate was rapidly taken up into the brain and was retained at high levels up to 20 min postadministration. The levels of [(14)C]phenylacetate in the cerebral cortex were about threefold higher than those of [(14)C]acetate at 1 min postinjection. Microinjection of fluorocitrate into the right striatum resulted in a significant decrease of the uptake of both [(14)C]phenylacetate and [(14)C]acetate into the right striatum. Radiochemical analysis confirmed the rapid hydrolysis of [(14)C]phenylacetate in the rat brain, with less than 20% of radioactivity representing unmetabolized [(14)C]phenylacetate at 1 min postinjection. These results suggest that [(14)C]phenylacetate is rapidly taken up into the brain and is hydrolyzed and converted to [(14)C]acetate. [(14)C]Phenylacetate may have the potential to serve as a tracer for the measurement of glial metabolism in an intact brain.