Introduction Intracellular Ca2+ handling in cardiac muscle plays an important role in cardiac contraction and relaxation. Recently, increased Ca2+ leak from sarcoplasmic reticulum (SR) through ryanodine receptor has been reported to decrease cardiac contraction. It has been also reported that there were more heart failure patients with preserved cardiac contractility than we expected in clinical field. Heart failure with preserved contractility presents diastolic dysfunction, which may be caused by impaired relaxation due to decreased Ca2+ uptake into SR. However, the relationship between Ca2+ uptake and leak in SR has not been fully investigated. In the present study, we investigated the relationship between Ca2+ leak from SR through ryanodine receptor and Ca2+ uptake into SR by SR Ca2+-ATPase (SERCA). Methods We used the mice hearts with overexpression of SERCA (SERCA-TG) or sarcolipin (SLN-TG) which suppresses SERCA activity. The hearts were quickly removed from mice anesthetized with sodium pentobarbital (300 mg/kg intraperitoneal administration). We used saponin-treated thin trabeculae and papillary muscles obtained from these mice hearts. Each SR function of these mice hearts was estimated by measuring the Ca2+ content in SR by using caffeine (50 mM) and fluo-3 when SR was loaded in the solution with various Ca2+ concentrations (pCa 8-5.6) and loading time (10-300 sec). Ca2+ transient and tension in twitch contraction of intact papillary muscles from mice were also measured. To estimate the effect of the beta-adrenergic stimulation on myocardium in SERCA-TG and SLN-TG, isoproterenol (100 nM) was applied to papillary muscles obtained from mice hearts. Results At short Ca2+ loading time, Ca2+ content in SR was larger in SERCA-TG (159%) and smaller in SLN-TG (50%) compared to each control myocardium. Ca2+ leak from SR in SERCA-TG and SLN-TG did not differ from each NTG. In SERCA-TG heart, the peak of Ca2+ transient increased and the time course of Ca2+ transient was accelerated. In SLN-TG heart, the peak of Ca2+ transient decreased and the time course of Ca2+ transient was slowed. In the papillary muscle preparation from control, isoproterenol increased Ca2+ transient and tension and accelerated time courses of both signals. In the papillary muscle preparation from SERCA-TG, Ca2+ transient and tension were not increased and time courses of both signals were not altered by isoproterenol treatment. In papillary muscles preparation from SLN-TG, Ca2+ transient and tension were significantly increased and time courses of both signals were significantly altered by isoproterenol treatment (e. g. Asahi et al. (2004)). Conclusion Selective modulation of Ca2+ uptake into SR influenced time course of Ca2+ transient, however, it did not influence Ca2+ leak from SR.