The mouse has been used extensively for generating transgenic animal models to study cardiovascular disease. Recently, a number of transgenic mouse models have been created to investigate the importance of sarcoplasmic reticulum (SR) Ca2+transport proteins in cardiac pathophysiology. However, the expression and regulation of cardiac SR Ca2+ATPase and other Ca2+transport proteins have not been studied in detail in the mouse. In this study, we used multiplex RNase mapping analysis to determine SERCA2, phospholamban (PLB), and Na+/Ca2+-exchanger (NCX-1) gene expression throughout mouse heart development and in hypo/hyperthyroid animals. Our results demonstrate that the expression of SERCA2 and PLB mRNA increase eight-fold from fetal to adult stages, indicating that SR function increases with heart development. In contrast, the expression of the Na+/Ca2+-exchanger gene is two-fold higher in fetal heart compared to adult. Our study also makes the important observation that in hypothyroidic hearts the NCX-1 mRNA and protein levels were upregulated, whereas the SERCA2 mRNA/protein levels were downregulated. In hyperthyroidic hearts, however, an opposite response was identified. These findings are important and point out that the expression of NCX-1 is regulated antithetically to that of SERCA2 during heart development and in response to alterations in thyroid hormone levels.