Junctin is one of the components of the ryanodine receptor Ca release channel complex in sarcoplasmic reticulum. To determine the role of acute alteration of junctin protein levels on cardiomyocyte contractility, we used adenoviral-mediated gene transfer techniques in adult rat cardiomyocytes. Acute downregulation of junctin by 40% resulted in significant increases in cell shortening, rate of contraction (+dL/dt), and rate of relaxation (-dL/dt). The alteration of contractile parameters was associated with increased Ca transient peak and accelerated Ca decay. However, all these contractile and Ca kinetic parameters were depressed significantly when junctin levels were upregulated by 60%. Importantly, there were no alterations in other Ca-cycling protein levels when junctin levels were either decreased or increased. These findings suggest that junctin plays a prominent role in cardiomyocyte Ca-cycling and contractility.