Proopiomelanocortin (POMC) is the common precursor of a variety of important endocrine peptides including ACTH. Transcription of the POMC gene is positively regulated by CRH through cAMP-responsive regions and is under negative feedback control by glucocorticoids which exert their inhibitory effect trough negative glucocorticoid responsive elements (nGRE). In vitro studies using the rat POMC promoter suggested that binding of the glucocorticoid receptor complex to a -63 bp binding site is correlated with repression of POMC gene transcription, and that specific mutations in this region abolish this effect. Impaired negative feedback regulation, though to a different degree, is a common feature of both corticotroph tumors (Cushing's disease) and extrapituitary ACTH producing tumors. We have analyzed the upstream promoter region of POMC gene from eleven patients with Cushing's disease, four of which had Nelson's syndrome, and from one patient with an ectopic ACTH syndrome secondary to a lung carcinoid for any possible mutations in the nGRE and/or cAMP-responsive sequences. DNA was purified from tumor tissue and was used as template for polymerase chain reaction (PCR). A segment between -371 and -19 bp of the POMC transcription start site was amplified and cloned into a plasmid vector. Sequencing was performed using the dideoxy chain termination procedure. Analysis of the 5'-flanking region revealed no defect in all tumors investigated. We conclude from our results that the defective glucocorticoid repression of POMC peptides production may be more likely due to aberrancies in other components of the complex transcriptional regulatory mechanism.