The molecular basis of Cushing's disease is not known. One of the most characteristic features of such tumors is their resistance to corticosteroid feedback at the pituitary level. We have hypothesized that abnormalities of the glucocorticoid receptor (GR) gene might play a role in the development of Cushing's disease via an increase in the relative production of the nonligand-binding splice variant of the GR, GR beta, known to exert dominant negative effects over the ligand-binding isoform, GR alpha. Alternatively, a change in overall GR expression, or mutations of some functional domains of the GR gene, might be involved in the pathogenesis of corticotroph tumors. We studied 22 tumors (17 pituitary ACTH-secreting tumors, 2 ectopic ACTH-producing tumors, 2 prolactinomas, and 1 nonfunctioning adenoma) and three normal pituitaries. RT-PCR was performed with primers specific to GR alpha and GR beta complementary DNA, followed by Southern blotting using an internal probe, and the ratio of the two bands quantitated by densitometry. We also assessed the overall expression of GR relative to the message of both the POMC gene and a housekeeping gene. Single-strand conformation polymorphism analysis of the DNA-binding domain and splice junction region of the gene was also performed. GR alpha messenger RNA was expressed at 37.3-fold +/- 5.7 (range, 32 to 46) excess, as compared with the GR beta subform. This pattern was observed both in the tumor samples and in the normal pituitaries used as controls. A majority of the ACTH-secreting tumors (16/19), including the ectopic secretors, showed variable but increased overall GR expression, whereas 3 tumors showed an expression approximately equivalent to the normal controls; however, no correlation was found between these two groups and the response to the high-dose dexamethasone test, nor was there any correlation with tumor histology. No mutations were found in any of the tumors by PCR-single-strand conformation polymorphism analysis. In conclusion, although both pituitary and ectopic ACTH-secreting tumors are at least partially glucocorticoid-resistant, no significant abnormalities in the relative expression of the two main GR subforms were observed in a series of such tumors. Additionally, mutations of regions critical to normal function of the receptor do not seem to be a frequent event in these tumors.