Aldosterone plays a key role in extracellular volume homeostasis by promoting sodium reabsorption in the kidney distal tubule. The accepted model for aldosterone action involves the binding of the hormone to the mineralocorticoid receptor (MR) in the cytoplasm and subsequent translocation of the aldosterone-MR complex to the nucleus, where it acts as a transcription factor. MR is also expressed in non-epithelial tissues such as the heart, where its physiological role is unclear. However, inappropriate MR activation leads to the development of cardiac fibrosis and renovascular disease. To improve our understanding of MR functional roles in the heart, we investigated the subcellular localization of the receptor in mouse left ventricle and in immortalized cardiac myocytes in the absence or presence of corticosteroids. Tissues were obtained from adrenal-intact or adrenalectomized (ADX) 8-week-old C57BL/6 mice. Preparative surgery was performed on mice anesthetized by intraperitoneal administration of 50 mg/kg ketamine and 1 mg/kg medetomidine. After the procedure, anaesthesia was reversed with 1 mg/kg atipamezole. Mice were kept for four days after ADX with free access to saline prior to harvesting the tissue. The procedures were approved by the local ethics committee and accorded with current national legislation. Three independent experiments with 3 mice in each group were performed. MR localization in the left ventricle was studied by cellular fractionation (1). MR subcellular localization was further tested by transient transfection of wild type or mutant MR in HL-1 cells derived from adult mouse cardiac myocytes (2). MR localization in HL-1 cells was studied by indirect immunofluorescence (3) and cellular fractionation (1). Three independent experiments were performed for each condition tested. Our results show that both endogenous MR in the left ventricle and transfected MR in HL-1 cells are detected exclusively as nuclear chromatin-bound factors. Depletion of corticosteroids by ADX in mice or by culturing HL-1 cells with charcoal-striped serum or serum-free medium did not affect MR chromatin binding. Deletion of nuclear localization signal 0 (NLS0) (1) rendered mutants exclusively cytoplasmic, while mutations in NLS1 partially redistributed MR to the cytoplasm. Taken together, our results suggest that MR is constitutively nuclear in mouse cardiac myocytes, independently of corticosteroids. The subcellular localization of the naïve receptor is mainly determined by NLS0. This stands in sharp contrast to the behaviour of MR in other cell types, where it appears evenly distributed over the nucleus and the cytoplasm. The differential behaviour of MR in cardiac myocytes might have important consequences for its function, both in genomic and non-genomic pathways.