Intramitochondrial glutamate removal through deamination may regulate renal ammoniagenesis from glutamine. However, little information is available to determine if there is a strong association between glutamine deamidation and the removal within mitochondria of the glutamate subsequently formed after deamidation. Using rat renal mitochondria, we found that ammonia production, glutamate appearance, and amide nitrogen disappearance were near equal aerobically and anaerobically, when no ADP-generating system was present. Whan an ADP-generating system was added (ATP alone, ATP + malonate, or ATP+ 2,4 DNP), more ammonia was formed aerobically from glutamine. Additionally, less glutamate appeared even though more amide nitrogen disappeared. Intramitochondrial concentrations of glutamate decreased. When pyruvate and α-ketoglutarate were added to the system, ammoniagenesis, deamidation, and deamination decreased; while glutamate built up in both the medium and mitochondria. In our mitochondrial system, we found a significantly positive correlation between glutamate deamination and glutamine deamidation, between glutamate accumulation and intramitochondrial glutamate concentrations; and a significantly negative correlation between glutamate deamination and glutamate accumulation, between glutamine deamidation and intramitochondrial glutamate concentrations, and between glutamate deamination and glutamate accumulation. We conclude that there is a biochemical relationship between glutamine deamidation and deamination of the glutamate subsequently formed. We propose that increased deamination lowers mitochondrial concentrations of glutamate and increases deamidation. In contrast, slowing deamination increases mitochondrial concentrations of glutamate and decreases deamidation.