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Background In patients with primary hyperaldosteronism, distinguishing between unilateral and bilateral adrenal hypersecretion is critical in assessing treatment options. Adrenal venous sampling (AVS) has been advocated by some to be the gold standard for localization of the responsible lesion, but there remains a lack of consensus for the criteria and the standardization of technique. Study Design We performed a retrospective study of 114 patients with a biochemical diagnosis of primary hyperaldosteronism who all underwent CT scan and AVS before and after corticotropin (ACTH) stimulation. Univariate and multivariate analyses were performed to determine what factors were associated with AVS lateralization, and which AVS values were the most accurate criteria for lateralization. Results Eighty-five patients underwent surgery at our institution for unilateral hyperaldosteronism. Of the 57 patients who demonstrated unilateral abnormalities on CT, AVS localized to the contralateral side in 5 patients and revealed bilateral hyperplasia in 6 patients. Of the 52 patients who showed bilateral disease on CT scan, 43 lateralized with AVS. The most accurate criterion on AVS for lateralization was the post-ACTH stimulation value. Factors associated with AVS lateralization included a low renin value, high plasma aldosterone-to plasma-renin ratio, and adrenal mass ≥ 3 cm on CT scan. Conclusions Because 50% of patients would have been inappropriately managed based on CT scan findings, patients with biochemical evidence of primary hyperaldosteronism and considering adrenalectomy should have AVS. The most accurate measurement for AVS lateralization was the post-ACTH stimulation value. Although several factors predict successful AVS lateralization, none are accurate enough to perform AVS selectively.