The human sarco/endoplasmic reticulum (ER) Ca(2+)ATPase 3 (SERCA3) gene gives rise to SERCA3a-3f isoforms, the latter inducing ER stress in vitro. Here, we first demonstrated the co-expression of SERCA3a, -3d and -3f proteins in the heart. Evidence for endogenous proteins was obtained by using isoform-specific antibodies including a new SERCA3d-specific antibody, and either Western blotting of protein lysates or immunoprecipitation of membrane proteins. An immunolocalization study of both left ventricle tissue and isolated cardiomyocytes showed a distinct compartmentalization of the SERCA3 isoforms, as a uniform distribution of SERCA3a was detected while -3d and -3f isoforms were observed around the nucleus and in close vicinity of plasma membrane, respectively. Second, we studied their expressions in failing hearts including mixed (MCM) (n=1) and idiopathic dilated (IDCM) cardiomyopathies (n=4). Compared with controls (n=5), similar expressions of SERCA3a and -3d mRNAs were observed in all patients. In contrast, SERCA3f mRNA was found to be up-regulated in failing hearts (125+/-7%). Remarkably, overexpression of SERCA3f paralleled an increase in ER stress markers including processing of X-box-binding protein-1 (XBP-1) mRNA (176+/-24%), and expression of XBP-1 protein and glucose-regulated protein (GRP)78 (232+/-21%). These findings revisit the human heart's Ca(2+)ATPase system and indicate that SERCA3f may account for the mechanism of ER stress in vivo in heart failure.