BACKGROUND As a consequence of the loss of liver function in chronic liver disease, increased levels of ammonia, manganese, and glutamine have been observed in the brain of hepatic encephalopathy patients. OBJECTIVE In the present study, we explored phosphate activated glutaminase (PAG) activity in mitochondrial enriched fractions under treatment with ammonia and manganese. METHODS We dissected out the brain cortex, striatum, and cerebellum of male Wistar rats 250-280 g weight; brain sections were pooled to obtain enriched mitochondrial fractions by differential centrifugation. Aliquots equivalent to 200 μg of protein were incubated with semi-log increasing concentrations of ammonia and/or manganese both as chloride salts (from 0 to 10 000 μM) and glutamine (4 mM) for 30 min. Then, the glutamate produced by the reaction was determined by HPLC coupled with fluorescence detection. CONCLUSIONS Both manganese and ammonia inhibited PAG in a concentration-dependent manner. Non-linear modeling was used to determine IC50 and IC20 for ammonia (120 μM) and manganese (2 mM). We found that PAG activity under the combination of IC20 of ammonia and manganese was equivalent to the sum of the effects of both substances, being PAG inhibition more pronounced in mitochondrial fractions from cerebellum. The PAG inhibition observed here could potentially explain a pathway for glutamine accumulation, by means of the inhibition of PAG activity as a consequence of increased concentrations of manganese and ammonia in the brain under liver damage conditions.