BACKGROUND: Adrenal vein sampling (AVS) is the preferred test for subtyping primary aldosteronism. However, the procedure is technically demanding and costly. In AVS it is common practice to take duplicate blood samples at each location. In this paper we explore whether a single sample procedure leads to a different conclusion concerning the location of adrenal aldosterone secretion than a duplicate sample procedure. METHODS: AVS procedures with duplicate measurements performed in our university medical center between 2005 and 2010 were evaluated retrospectively. We compared the conclusions regarding selectivity and lateralization based on the first sample taken (A) to the conclusions based on the average of duplicate samples (AB). We also calculated the number needed to be sampled in duplicate to prevent one misclassification. RESULTS: Ninety-six AVS procedures of 82 patients were included. The concordance in AVS conclusions between samples A and AB was 98-100%, depending on the criteria used for selectivity and lateralization. With permissive and strict criteria the number needed to be sampled in duplicate were infinite and 48, respectively. CONCLUSIONS: The incremental benefit of duplicate sampling compared to single sampling is low. Therefore, in the case of technical difficulties during AVS, conclusions can also be reliably drawn from a single blood sample.