The reproducibility of adrenal vein sampling(AVS) is unknown.Determine reproducibility of biochemical results and diagnostic lateralization in patients undergoing repeat AVS.Retrospective chart review of single-center, single-operator AVS procedures.Tertiary care center.Patients with confirmed PA undergoing repeat AVS due to concerns about technical success or discordant diagnostic results.Simultaneous AVS by an experienced operator using a consistent protocol of both pre-and post-cosyntropin infusion.Among successfully catheterized adrenal veins(selectivity index ≥ 2) the correlation of the adrenalaldosterone/cortisol ratio between the first and second AVS. Secondary outcome measure was diagnostic agreement in repeat AVS lateralization(lateralization index ≥ 3).There were 46 sets of AVS from 23 patients, median 3 months apart. There was moderate correlation in aldosterone/cortisol ratios in adrenal veins and IVC(Spearman r = 0.49-0.59, p<0.05) pre-cosyntropin. Post-cosyntropin, the correlation was better(Spearman r=0.67-0.76, p<0.05). In technically successful AVS, there was moderate correlation between the repeated lateralization indices(Spearman r=0.53, p<0.05). In 15 patients where repeat AVS was done due to apparent lateralization discordance with CT imaging, the final diagnosis was the same in the second AVS procedure. Initial failed AVS was successful 75% of the time upon repeat attempt.Repeat AVS was feasible and usually successful when an initial attempt failed. There was modest correlation between individual repeat adrenal aldosterone/cortisol ratios and lateralization indices when AVS was done twice. Final lateralization diagnosis was identical in all cases. This demonstrates that AVS is a reliable and reproducible localizing test in PA.