When rat brain synaptic and non-synaptic mitochondria were incubated with [14C]glutamine, [14C]glutamate was rapidly released to the incubation medium, and the release was stimulated by phosphate, whereas [14C]glutamate accumulated very slowly in the mitochondria and independently of the addition of phosphate. The specific activity of [14C]glutamate (dpm.nmol glutamate-1) in the incubation medium quickly reached the level of added [14C]glutamine, but the specific activity of [14C]glutamate in the mitochondria was found to be only 10-15% of that level. This indicates that glutamine-derived glutamate was released directly to the incubation medium, without being mixed with a general pool of endogenous glutamate in the mitochondria. Furthermore, there was no correlation between rate of glutamine hydrolysis and the uptake of glutamine into the mitochondria, as measured by the uptake of [3H]glutamine and glutamine induced mitochondrial swelling when calcium plus phosphate or asparagine were added. Glutamine hydrolysis was also not stimulated by partial disruption of the mitochondria following sonication, which should be expected if the rate of glutamine hydrolysis were limited by glutamine uptake. In addition, glutamine hydrolysis was strongly inhibited by mersalyl which is known to be impermeable to the inner mitochondrial membrane. Moreover, it is indicated that the enzyme was not an integral membrane protein. Thus, following fractionation of a Triton X-114 extract of brain synaptosomes, a major fraction of both the protein, as measured by immunoblot technique, and the enzyme activity were detected in the water phase. Our results therefore indicate that the whole molecule of phosphate activated glutaminase is externally localized in the inner mitochondrial membrane.