Abstract Primary aldosteronism (PA) is the most common cause of endocrine hypertension. PA subtypes include bilateral hyperplasia and unilateral PA, typically aldosterone-producing adenomas. Adrenal venous (AV) sampling (AVS) is a key step to define PA subtype and guide PA management. According current PA guidelines, most PA patients should undergo AVS, which is a challenging procedure, especially in terms of successfully cannulating the right AV. The aim of this study was to report a single tertiary center experience with AVS in PA patients. We retrospectively evaluated 84 AVS from 1984 to 2019. Sequential AVS was performed by an experienced interventional radiologist. AV and inferior vena cava (IVC) samples were obtained under cosyntropin continuous infusion. Successful catheterization was defined by a selectivity index SI= AV/IVC cortisol (C) concentrations ≥5. Unilateral disease was defined by a lateralization index LI= aldosterone (A)/C ratio in the dominant AV divided by A/C in the non-dominant AV ≥4. The relative aldosterone secretion index (RASI= A/C ratio in AV divided by A/C in IVC) was calculated in each side. A RASI <1 was defined as contralateral suppression (CS). In patients with unsuccessful AV catheterization (mostly right AV) or undetermined LI (3-4), CS was used to indicate adrenalectomy. The biochemical cure of PA after adrenalectomy was defined as the gold standard parameter to confirm unilateral disease. Successful bilateral AV catheterization was achieved in 75% of the cases. After 2015, the use of intra-procedural rapid cortisol assay improved angiographer experience and increased AVS successful rate from 52 to 80%. LI revealed unilateral and bilateral aldosterone excess in 68 and 32% of the cases, respectively. A LI ≥4 had a sensitivity of 100% and specificity of 98% to define unilateral PA among patients with successful catheterization. In addition, RASI in the non-dominant AV was significantly lower in unilateral PA according the LI when compared to bilateral cases 0.12 (0.03 to 1.18) vs. 1.1 (0.04 to 4.56), p= 0.0001. RASI in the non-dominant AV was inversely correlated with LI (r= -0.81, p= 0.0001). A CS index ≤0.5 had a high sensitivity (90%) and specificity (94%) to define unilateral aldosterone excess. In conclusion, the LI is the most valuable parameter in AVS for PA subtyping. Additionally, CS (cut-off of 0.5) is very useful to define lateralization and can be used in cases of borderline LI or unsuccessful AV catheterization.CAPES Grant to Freitas TC.