Glucocorticoid hormones regulate the transcription of nuclear genes by way of their cognate receptors. In addition, these hormones also modulate mitochondrial gene transcription by mechanisms which are as yet poorly understood. Using immunofluorescence labeling and confocal laser scanning microscopy we show that the glucocorticoid receptor of HeLa and Hep-2 cells is specifically enriched at the sites of the mitochondria which were visualized by labeling with the vital dye CMX and antibodies against cytochrome oxidase subunit I. Immunogold electron microscopy demonstrated that the receptor was located within the inner space of the mitochondria. Immunoblotting experiments also revealed the presence of glucocorticoid receptor in mitochondria isolated from HeLa and Hep-2 cells. Finally, living HeLa cells expressing green fluorescent-glucocorticoid receptor fusion protein revealed a distinct mitochondrial GFP fluorescence. Our results support the concept of a receptor-mediated direct action of steroid hormones on mitochondrial gene transcription.