The metabolic process of glutamate synthesis in Saccharomyces cerevisiae is governed by a functional model that assumes extramitochondrial localization of glutamate dehydrogenase (GDH). In S. cerevisiae, GDH exists as three isozymes: Gdh1p and Gdh3p, which are NADP-dependent, and Gdh2p, which is NAD-dependent. Previous work has suggested nuclear and/or cytosolic localization, but definitive data are lacking. We attempted to determine the subcellular localization of the three GDH isozymes by enzymatic activity. Crude mitochondrial and extramitochondrial fractions were isolated from wild-type and mutant (gdh3Δ) cells by subcellular fractionation. Spectrophotometric measurement of NADP-GDH and NAD-GDH activity suggested extramitochondrial localization for Gdh1p and Gdh2p. Additionally, NADP-GDH activity was lower in gdh3Δ extramitochondrial fractions as compared to wild-type. However, this trend was not observed in mitochondrial fractions, suggesting extramitochondrial localization for Gdh3p. Since mammalian GDH is primarily mitochondrial, our findings have implications for the use of S. cerevisiae in modeling glutamate metabolism.