Introduction: Cardiac contraction results from a brief increase in cytosolic calcium (Ca) concentration (systolic Ca transient). The majority of this increase is due to release of Ca from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). For relaxation to occur, the RyRs close and Ca is removed from the cytoplasm, largely by pumping Ca back into the SR via the sarco-endoplasmic reticulum Ca ATPase (SERCA). If the RyR becomes leaky then the resulting extra efflux of Ca from the SR will reduce the effects of SERCA-mediated Ca uptake and decrease SR Ca content and thence the amplitude of the Ca transient. Previous work has also shown that increasing RyR leak slows the rate of decay of the Ca transient and this may contribute to impaired relaxation in heart failure [1]. The aim of this study was to characterize the precise details of this slowing of decay of the Ca transient. Methods: Single rat ventricular myocytes were voltage clamped using the perforated patch clamp technique. 100 ms duration pulses from -40 to 0 mV were applied at 0.5 Hz. Cytosolic Ca concentration was measured using Fluo-4 AM. SR Ca leak was induced using 1 mM caffeine. Statistical analysis was performed using the t-test. Results: Application of caffeine decreased the amplitude of the Ca transient (to 45% of control) and its rate constant of decay (to 75%). In 3 out of 11 cells the decay of the Ca transient was clearly biphasic with an initial rapid phase of decay followed by a slower one. This biphasic decay was seen much more frequently (12 out of 15 cells) if caffeine was applied in the presence of isoprenaline (1 µM) as shown in Fig. 1 (A-raw trace and B- normalised). In isoprenaline the Ca transient decays as a single exponential with a rate constant of decay of 18.1±1.8 s-1. The addition of caffeine results in bi-exponential decay with the rate constant of the fast phase (16.5±2.4 s-1) being the same (p=0.61) as in the absence of caffeine. Thus, in the normalized traces (B), the initial fast phase superimposes on that in isoprenaline alone. The slow phase has a rate constant of 1.3±0.3 s-1. We hypothesise that the initial rapid phase of decay is a consequence of the SR Ca content and therefore leak being reduced following systolic release. Only when SR Ca content begins to increase does the leak become sufficient to significantly oppose SERCA. In 6 cells showing biphasic decay in caffeine plus isoprenaline we investigated the effect of partial inhibition of SERCA and found that thapsigargin reduced the amplitude and rate constant of decay of the fast component . Conclusions: We conclude that Ca leak from the SR can lead to biphasic decay of the Ca transient because the effect of leak is greater at later times when the SR Ca content is increased. These results will be relevant to relaxation in heart failure.