The aim of our study was to assess the frequency of 21-hydroxylase deficiency, a cause of congenital adrenal hyperplasia (CAH), in incidentally discovered asymptomatic adrenal masses (incidentalomas) and to compare the prevalence of this enzymatic disorder in monolateral (M) and bilateral (B) forms. Twenty-seven patients with incidentalomas (12 M and 15 B) and 16 sex and age-matched controls (C) received synthetic adrenocorticotropin (ACTH, 250 micrograms i.v.). Plasma 17-OHprogesterone (17-OHP) and cortisol were collected in basal conditions and after 30, 60, 90 minutes. Basal plasma 17-OHP in C [1.25 +/- 0.15 (0.61) ng/ml, mean +/- SE (SD)] was not significantly different from that in patients with M [0.85 +/- 0.13 (0.44) ng/ml] or B [0.94 +/- 0.23 (0.90) ng/ml] incidentalomas. After ACTH, 17-OHP levels significantly (p < 0.05) increased in C, in M and B incidentalomas. However, the rise in plasma 17-OHP in C both in terms of peak [2.5 +/- 0.28 (1.1) ng/ml] and of AUC values [174 +/- 16 (64) ng/ml/min] was significantly lower than that observed in M [peak 6.32 +/- 1.66 (5.7) ng/ml, p < 0.01; AUC 410 +/- 111 (385.5) ng/ml/min, p < 0.01] and in B [peak 8.84 +/- 1.98 (7.65) ng/ml, p < 0.001; AUC 613 +/- 149 (579.3), ng/ml/min, p < 0.001] incidentalomas. Individual data indicated that while 17-OHP response to ACTH in C never reached 5 ng/ml (cut-off for normal response), 16 out of 27 patients with incidentalomas (59.2%) exceeded this value. Moreover, the abnormal response was more frequently observed in B (66.6%) than in M (50%) incidentalomas. Basal and stimulated plasma cortisol did not differ among the three groups. In conclusion, our data indicate that in adrenal incidentalomas the endocrine pattern of 21-hydroxylase deficiency is very common and that this enzymatic defect is more frequent in bilateral than in monolateral lesions.