Accurate localization of aldosterone-producing adenoma (APA) is essential for the treatment of primary aldosteronism (PA). In order to confirm the clinical usefulness of adrenal venous sampling (AVS), we retrospectively studied 87 cases of PA in whom AVS was conducted. We collected right and left adrenal venous effluents simultaneously before and after adrenocorticotropic hormone (ACTH) stimulation for measurements of aldosterone concentration (A) and cortisol concentration (C). Based on AVS results, we judged 66 cases as having unilateral aldosterone hypersecretion and the remaining 21 cases as having no apparent laterality. Of the above 66 subjects, 61 underwent laparoscopic removal of the adrenal gland through a retroperitoneal approach. The presence of APA was histopathologically confirmed, and blood pressure decreased significantly with normalization of plasma aldosterone concentration (PAC) in all cases. The receiver operator characteristics (ROC) curve analysis between the operated and no-apparent-laterality groups revealed that the ratio of A/C on the higher side to A/C on the lower side (A/C ratio) after ACTH stimulation is a useful index, with a cutoff value of 2.6, a sensitivity of 0.98 and a specificity of 1.0. The ROC curve analysis between the APA side and contralateral side within the operated patients revealed that the cutoff value of A was 1,340 ng/dL, with a sensitivity of 0.92 and a specificity of 1.00. Our results indicate the usefulness of simultaneous AVS and ACTH stimulation for localizing APA.