Context: 18-Oxocortisol (18-oxoF) is a derivative of cortisol (F) that is produced by aldosterone synthase (CYP11B2). The potential for this steroid as a biomarker for differentiating patients with aldosterone-producing adenoma (APA) from those with idiopathic hyperaldosteronism (IHA) has not been examined. Objectives: We measured 18-oxoF, aldosterone, and F in plasma from adrenal vein sampling (AVS) of patients with primary aldosteronism.Wecompared 18-oxoF levels and 18-oxoF/F ratios for their potential to differentiate APA from IHA. Design, Setting, and Subjects: This study measured 18-oxoF, F, and aldosterone in AVS obtained from patients with unilateral APA (14 cases) or bilateral IHA (seven cases, 14 samples total) using liquid chromatography-tandem mass spectrometry and RIA analyses. Results: The levels of 18-oxoF and the ratios of 18-oxoF/F, before and after ACTH stimulation, were significantly higher in blood-draining APA than in those from the contralateral adrenal glands and from adrenal glands with IHA. Conclusions: The 18-oxoF levels and ratios of 18-oxoF/F in AVS samples can be a clinically useful biomarker for differentiating APA from IHA and for determining the localization or lateralization of APA in patients with primary aldosteronism.