The pre-B lymphocyte protein 3 (VPREB3) is expressed during B-cell differentiation and in subsets of mature B-lymphocytes and is mainly found in bone marrow and lymphoid tissue germinative centers. So far, its function in B-cells remains to be clarified. The mRNA of VPREB3 was previously detected in aldosterone-producing adenomas (APA), however further information about this protein in human adrenocortical cells and tissues is currently unavailable. Therefore, in the present study we for the first time investigate the protein expression of VPREB3 in human adrenocortical tissues. In addition, we approach the previously suggested similarities in expression patterns of aldosterone-producing cells and Purkinje neurons. Immunohistochemical analysis of VPREB3 was performed in 13 non-pathological adrenals (NA), 6 adrenal glands with idiopathic hyperaldosteronism (IHA), 18 APA, 5 cortisol-producing adenomas (CPA) and 5 non-pathological human cerebellum specimens. The mRNA levels of VPREB3, steroidogenic-enzymes and other aldosterone biosynthesis markers were detected in 53 APA samples using real-time RT-PCR (qPCR) and compared to the clinical data of APA patients. In our results, the VPREB3 protein was diffusely detected in APA, partially or weakly detected in CPA, and immunolocalized in the zona glomerulosa of NA and IHA, as well as in the cytoplasm of cerebellar Purkinje cells. In APA, VPREB3 mRNA levels were significantly correlated to plasma aldosterone (P=0.026; R=0.30), KCNJ5 mutations (P=0.0061; mutated 34:19 wild-type), CYP11B2 (P<0.0001; R=0.65), Purkinje cell protein 4 (PCP4; P<0.0001; R=0.53) and voltage-dependent calcium channels CaV1.3 (P=0.023; R=0.31) and CaV3.2 (P=0.0019; R=0.42). Based on our data, we hypothesize a possible role for VPREB3 in aldosterone biosynthesis, and present ideas for future functional studies.