Adrenal vein sampling (AVS) is crucial for accurate lateralization of aldosterone excess but it is technically challenging due to the difficulty of adrenal vein cannulation. The use of adrenocorticotropic hormone (ACTH) to improve cannulation success is controversial and can lead to discordant lateralization outcomes.To evaluate the utility of ACTH in two centres with different levels of AVS expertise and formulate a strategy for interpreting discordant results.A retrospective cross-sectional analysis of AVS results and post-operative patient outcomes.Two large tertiary hospitals with harmonized AVS protocols where adrenal venous samples are collected both before and after ACTH stimulation.Cannulation success (measured by selectivity index, SI), lateralization (measured by lateralization index, LI) and post-operative biochemical cure.Number of AVS procedures judged to have successful bilateral adrenal vein cannulation increased from 53% pre- to 73% post-ACTH. The increase in cannulation success was significantly higher in centre where AVS was performed by multiple radiologists with a lower basal success rate. In both centres, the proportion of cases deemed to display lateralization significantly decreased with the use of ACTH (70% pre- to 52% post-ACTH). Based on post-operative outcomes of patients with discordant results who underwent unilateral adrenalectomy, the combination of LI > 3 pre-ACTH and LI > 2 post-ACTH was predictive of a biochemical cure.ACTH can increase the rate of cannulation success during AVS at the expense of reduced lateralization. The criteria for lateralization should be carefully determined based on local data when ACTH is used.