Glucocorticoid hormones regulate the transcription of nuclear genes by way of their receptors. In addition, these hormones modulate mitochondrial gene transcription by mechanisms that remain poorly understood. Using immunofluorescence labeling in isolated Muller and photoreceptor cells and in intact salamander retina, we found that the glucocorticoid receptor (GR) is localized in both cell types. Confocal laser scanning microscopy and double staining with cytochrome oxidase (COX) showed that GR is localized in the mitochondria of Muller cells, but not in the mitochondria of photoreceptors. GR also colocalizes with glutamine synthetase (GS) in the cytoplasm of Muller cells. GR is also localized in the microvilli of the distal process of Muller cells and in the synaptic terminal of photoreceptors. Pre-incubation of Muller cells with 1 microM dexamethasone (DEX) for 7 h led to greater than 50% inhibition of the glutamate-induced increase in mitochondrial NADH. This late effect of glucocorticoids on glutamate metabolism could be ascribed, in part, to a direct action of steroid hormones on mitochondrial metabolism.