Objective: In cardiac myocytes, nuclear [Ca] plays an important role in the regulation of transcription and thus the development of hypertrophy and heart failure. It is unclear though how nuclear [Ca] is regulated. Therefore, we studied nucleus and nuclear envelope (NE) morphology and the expression of Ca-regulating proteins in nuclei isolated from human myocardium. Methods: Nuclei were isolated from atrial and ventricular myocardium of non-failing and failing human hearts. Expression of proteins was determined using immunoblots. Morphology of isolated nuclei was evaluated using transmitted light microscopy; NE Ca stores were visualized by confocal microscopy and staining with the low affinity Ca dye Mag-Fluo-4/AM (10mM, 45min). Results: Purity of the nuclear fraction was confirmed by concentration of the nuclear markers nucleolin and nucleoporin 62 (Nup62), the transcriptional regulators GATA4 and Nkx2.5 (a cardiomyocyte-specific protein), as well as the absence of the cytoplasmic marker GAPDH. In light microscopy, nuclei were identified by staining with the nucleic acid dye Syto-16 (0.1mM). Nuclei isolated from atrial and ventricular myocardium were ellipsoid in shape and measured 9.3+/­0.5mm and 11.7+/­0.6mm, respectively, in length and 4.4+/­0.3mm and 5.3+/­0.3mm, respectively, in width (n=159 vs n=327). Ca-regulating protein expression in the nuclear fraction was normalized to Nup62 and included calsequestrin, phospholamban, SERCA2a, Na/Ca exchanger, ryanodine receptor, and IP3 receptor in all four chambers. Staining with Mag-Fluo-4 revealed a prominent NE and tubular structures (i.e. nucleoplasmic reticulum) traversing the nucleus, indicating high Ca concentrations in these compartments. Conclusions: Nuclei isolated from human myocardium contain a functional NE and a nucleoplasmic reticulum as Ca storage compartment and express the entire set of Ca-regulating proteins required for active uptake and release of Ca. Thus, cardiac nuclei may be capable of autonomous Ca regulation.