Objective: Absence of a late-night cortisol nadir is a consistent biochemical abnormality in patients with cortisol-producing adenoma. We evaluated the abnormality of late-night urinary free cortisol to creatinine ratio (late-night UFCCR) in patients with subclinical Cushing's syndrome (SCS). Methods: Fifty-eight patients with incidentally detected adrenocortical adenomas [SCS: 9; nonfunctioning adenoma (NF): 49] were enrolled as subjects. Values measured in all patients were urinary free cortisol accumulated between 9:00 p.m. and 11:00 p.m. (late-night UFCCR), serum cortisol at 11:00 p.m. (midnight serum cortisol: MSC), serum cortisol after 1-mg overnight dexamethasone suppression test (1 mg-DST) and 24-h urinary free cortisol (UFC). Results: Median late-night UFCCR value in SCS was significantly higher than that in NF (P < 0.001). Significant correlations were observed between late-night UFCCR and each of serum cortisol after 1 mg-DST and MSC (r = 0.537, P < 0.001 and r = 0.556, P < 0.001, respectively). There was no significant correlation between serum cortisol after 1 mg-DST and 24-h UFC (r = 0.211, P = 0.112). In receiver operating characteristic analysis for diagnosis of SCS, the areas under the curves of latenight UFCCR and 24-h UFC were 0.937 (95% confidence interval 0.865-1.008) and 0.726 (0.874-0.999), respectively. Late-night UFCCR cut-off value of 4.9 nmol/μmol Cre showed a sensitivity of 100% and a specificity of 76.6%. Conclusion: Patients with SCS showed higher late-night UFCCR values than those with NF. Late-night UFCCR was significantly correlated with autonomous cortisol production findings. Diagnostic performance of late-night UFCCR was superior to 24-h UFC. These results suggest that late-night UFCCR might represent one of the simple and reliable tests for SCS diagnosis.