The diagnosis of Cushing's syndrome (CS) is often a challenge. Recently, the determination of late night salivary cortisol levels has been reported to be a sensitive and convenient screening test for CS. However, no studies have included a comparison with other screening tests in a setting more closely resembling clinical practice, i.e. few patients with CS to be distinguished from patients with pseudo-Cushing states (PC), including the large population of obese patients. The aim of this study was to compare the diagnostic performance of midnight salivary cortisol (MSC) measurement with that of midnight serum cortisol (MNC) and urinary free cortisol (UFC) in differentiating 41 patients with CS from 33 with PC, 199 with simple obesity, and 27 healthy normal weight volunteers. Three patients with CS had MSC levels lower than the cut-off point derived from receiver operator characteristic analysis (9.7 nmol/liter), yielding a sensitivity for this parameter of 92.7%. In the whole study population, no statistically significant differences in terms of sensitivity, specificity, diagnostic accuracy, and predictive values were observed among tests. In particular, the overall diagnostic accuracy for MSC (93%; 95% confidence interval, 90.1-95.9%) was similar to those of UFC (95.3%; 94.1-96.5%) and MNC (95.7%; 93.4-98%; both P = NS). The diagnostic performance of MSC was superimposable to that of MNC also within the area of overlap in UFC values (< or =569 nmol/24 h) between CS and PC. In conclusion, MSC measurement can be recommended as a first-line test for CS in both low risk (simple obesity) and high-risk (i.e. PC) patients. Given its convenience, this procedure can be added to tests traditionally used for this purpose, such as UFC and MNC.