The study aimed to retrospectively evaluate the reliability of the diagnostic and location tests in Cushing's Syndrome (CS). Eighty-seven patients diagnosed with CS between 1995 and 2007 by Endocrinology Metabolism Department of Cerrahpasa Medical School were included in the study. The control group consisted of 91 patients who presented to the outpatient clinic because of obesity. The diagnostic tests were as follows: 1 mg dexamethasone suppression test (DST), 24-h urinary free cortisol (UFC), midnight cortisol level (MCL), ACTH level and overnight 8 mg DST. The sensitivity and specificity of UFC were 81 and 66 % respectively for the cut-off point of 50 mu g/day, whereas they were 64 and 76 % respectively for the cut-off point of 100 mu g/day. For the cut-off value of 1.8/mu g/dL for MCL and 1 mDST, the sensitivity rates were 100 and 98 %, while the specificity rates were 88 and 33 %, respectively. Among the location tests, the sensitivity and specificity of ACTH under 10 pg/mL for adrenal CS were 92 and 94 % respectively. The sensitivity and specificity of ACTH higher than 30 pg/mL for ACTH-dependent CS were 69 and 100 % respectively. The sensitivity rates of 8 mg DST for 50 and 60 % suppressions were 83 and 79 % respectively, whereas the specificity rates were 75 and 88 % respectively. 1 mg DST (cut-off < 1.8 mu g/dL) and UFC (50 mu g/24 h) are appropriate tests for screening CS. Overnight 8 mg DST with 60 % suppression for Cushing's Disease (CD) and ACTH levels < 10 pg/mL for adrenal CS, ACTH levels > 30 pg/mL for ACTH dependency were identified as the best tests for the differential diagnosis of the subtypes.