Regulation by ammonia of phosphate-dependent glutaminase in isolated rat-liver mitochondria was studied at pH values near the cytosolic pH of 7.0. 1. Glutaminase activity, both in the absence and presence of bicarbonate, was completely dependent on the presence of ammonia. 2. Glutaminase activity, both in the absence and presence of bicarbonate, was strongly depressed by decreasing the pH of the incubation medium from 7.0 to 6.8 when the ammonia concentration was below 0.5 mM. 3. Bicarbonate stimulated glutaminase activity only in the presence of low concentrations of ammonia. 4. The data indicate that the reported inhibition of glutamine degradation in the perfused liver at low pH [e.g. Häussinger et al. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 995-1001] is due to a decreased affinity of glutaminase for ammonia.