Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent adrenal Cushing's syndrome (CS), which is often associated with Carney complex (CNC). We have recently described a paradoxical increase in cortisol excretion after dexamethasone administration in most patients with PPNAD. In the present study we investigated the hypothesis that this phenomenon is due to a primary abnormality of the tissues affected by PPNAD, rather than a defect of the patients' hypothalamic-pituitary-adrenal axis; as such it should be replicated in vitro by adrenal slices exposed directly to dexamethasone. We were able to study adrenal tissues from eight patients with CS caused by PPNAD; two patients were also studied in vivo according to a protocol first described in ACTH-independent macronodular adrenal hyperplasia (AIMAH) for the clinical detection of aberrant hormone receptor expression. Their DNA has been previously screened for inactivating mutations of the PRKAR1A gene, the most frequent molecular defect leading to PPNAD and/or CNC. We also investigated whether glucocorticoid receptor (GR) expression underlies paradoxical dexamethasone responses in PPNAD by immunohistochemistry and semiquantitative PCR, and we correlated GR expression with that of other markers for PPNAD (e.g. synaptophysin). Indeed, we demonstrated that dexamethasone induced cortisol secretion in vitro in five of these tumors; no such increase was seen in adenomatous or AIMAH tissues that were treated in the same manner. GR mRNA was expressed, and GR immunoreactivity was detected in PPNAD nodular cells. Staining for GR was not seen in surrounding cortical cells, and hence, it correlated with synaptophysin, which also stains PPNAD in a similar manner. In normal adrenal tissue, GR was detected mostly in medullary areas, whereas GR immunoreactivity was weak in adenomatous and AIMAH tissues. We conclude that 1) dexamethasone produces an increase in glucocorticoid synthesis by PPNAD adrenal slices in vitro, suggesting a direct effect on adrenocortical tissue, and 2) this phenomenon is accompanied by increased expression of the GR in PPNAD nodules. PPNAD and/or CNC patients with and without mutations leading to protein kinase A activation demonstrated in vitro and/or in vivo paradoxical dexamethasone responses and GR expression, indicating that PRKAR1A alterations are not necessary for these phenomena.