The source of nitrogen (N) for the de novo synthesis of brain glutamate, glutamine and GABA remains controversial. Because leucine is readily transported into the brain and the brain contains high activities of branched‐chain aminotransferase (BCAT), we hypothesized that leucine is the predominant N‐precursor for brain glutamate synthesis. Conscious and unstressed rats administered with [U‐¹³C] and/or [¹⁵N]leucine as additions to the diet were killed at 0–9 h of continuous feeding. Plasma and brain leucine equilibrated rapidly and the brain leucine‐N turnover was more than 100%/min. The isotopic dilution of [U‐¹³C]leucine (brain/plasma ratio 0.61 ± 0.06) and [¹⁵N]leucine (0.23 ± 0.06) differed markedly, suggesting that 15% of cerebral leucine‐N turnover derived from proteolysis and 62% from leucine synthesis via reverse transamination. The rate of glutamate synthesis from leucine was 5 µmol/g/h and at least 50% of glutamate‐N originally derived from leucine. The enrichment of [5‐¹⁵N]glutamine was higher than [¹⁵N]ammonia in the brain, indicating glial ammonia generation from leucine via glutamate. The enrichment of [¹⁵N]GABA, [¹⁵N]aspartate, [¹⁵N]glutamate greater than [2‐¹⁵N]glutamine suggests direct incorporation of leucine‐N into both glial and neuronal glutamate. These findings provide a new insight for the role of leucine as N‐carrier from the plasma pool and within the cerebral compartments.