Objective: To evaluate the diagnostic performance of four different tests in order to differentiate between Cushing's disease (CD) and pseudo-Cushing's syndrome (PCS). Methods: In this prospective study, a total of 73 patients with clinical features of hypercortisolism and insufficient suppression of serum cortisol after 1 mg overnight dexamethasone and/or an elevated excretion of cortisol in 24-h urine samples were included. The circadian rhythm of serum cortisol levels as well as midnight serum cortisol (MserC) levels were assessed in all 73 patients. Late-night salivary cortisol (LNSC) concentrations were obtained in 44 patients. The dexamethasone-CRH (Dex-CRH) test was performed in 54 patients. Results: Fifty-three patients were diagnosed with CD and subsequently treated. Twenty patients were classified as having PSC. Serum cortisol circadian rhythm: the diurnal rhythmicity of cortisol secretion was retained in PCS. A cortisol midnight:morning ratio of O0.67 is highly suggestive of CD (positive predictive value (PPV) 100% and negative predictive value (NPV) 73%). MserC concentration O243 nmol/l has a PPV of 98% in predicting true CD (NPV 95%). LNSC level O9.3 nmol/l predicted CD in 94% of patients (NPV 100%). Dex-CRH test: after 2 days of dexamethasone suppression, a CRH-stimulated cortisol level O87 nmol/l (TZ15 min) resulted in a PPV of 100% and an NPV of 90%. Conclusion: The Dex-CRH test as well as a single measurement of cortisol in serum or saliva at late (mid-) night demonstrated high diagnostic accuracy in differentiating PCS from true CD.