The three isozymes of isocitrate dehydrogenase in Saccharomyces cerevisiae differ in subunit structure, subcellular location, and cofactor specificity. The two mitochondrial isozymes, IDH and IDP1, are NAD- and NADP-specific, respectively. Several lines of evidence presented here confirm the importance of IDH to respiratory processes. Expression of IDH RNA and protein is low with growth on glucose and is elevated with growth on non-fermentable carbon sources, a pattern of expression similar to that seen for other tricarboxylic acid cycle enzymes. In addition, a disruption mutant lacking IDH activity exhibits reduced growth rates on non-fermentable carbon sources, and mitochondria isolated from this mutant are incapable of respiration with added citrate. In contrast, IDP1 expression levels appear to be unresponsive to carbon source, and an IDP1 disruption mutant is not significantly impaired for growth or mitochondrial respiration. These results strongly suggest that IDP1 is incapable of participating in tricarboxylic acid cycle-based respiration despite its mitochondrial location. Analysis of the IDP1 and IDH disruption mutants for glutamate auxotrophy showed that either enzyme can contribute alpha-ketoglutarate for endogenous glutamate synthesis. IDH expression levels were found to be repressed in response to added glutamate during growth on glucose, while IDP1 expression levels remained unchanged. A double mutant lacking both IDP1 and IDH activities proved to be auxotrophic for glutamate during growth on glucose, but was capable of growth independent of added glutamate on non-fermentable carbon sources. These results suggest that the cytosolic NADP-specific IDP2 isozyme may provide alpha-ketoglutarate both for tricarboxylic acid cycle carbon flux and for cytosolic glutamate synthesis during growth on non-fermentable carbon sources in the absence of mitochondrial isocitrate dehydrogenase activity.