Glutamate has been implicated in signal transmission between sensory hair cells and afferent fibers in the inner ear. However, the mechanisms responsible for glutamate replenishment in these cells are not known. Here we provide evidence that phosphate activated glutaminase, which is thought to be the predominant glutamate-synthesizing enzyme in the brain, is concentrated in all types of hair cell in the organ of Corti and vestibular epithelium. By use of two different antibodies (directed to the N and C terminus, respectively) it was shown that glutaminase is largely restricted to mitochondria and that part of the enzyme pool is associated with the inner membrane of this organelle. Quantitative analysis of immunogold labelled Lowicryl sections revealed that the level of glutaminase immunoreactivity in mitochondria of supporting cells is less than 15% of that in hair cell mitochondria. Using triple labelling for glutaminase, glutamate, and glutamine, evidence was provided of a positive correlation between the glutamate/glutamine ratio and the level of glutaminase immunoreactivity, suggesting that the glutaminase antibodies identify a functional enzyme pool. Our results strengthen the idea that glutamate is a hair cell transmitter and indicate that the sensory epithelia in the inner ear show a metabolic compartmentation analogous to that in the brain.