Introduction: Phosphodiesterase 5A inhibition with sildenafil (Sil) improves cardiac function and functional indices in heart failure (HF)1. In addition, acute application of Sil in animal models of myocardial infarction has direct cardioprotective and antiarrhythmic effects2. Sildenafil reduces L-type Ca2+ current (ICa,L) 3 and attenuates adrenergically-driven inotropism4, yet effects on myocyte calcium handling are largely undetermined. Our aim was to establish whether sildenafil exerts antiarrhythmic and negatively inotropic effects through alterations in Ca2+ handling. Methods: Isolated adult rat ventricular myocytes (ARVMs) were voltage clamped using whole-cell or perforated patch techniques, and Ca2+ fluorescence measured using the indicator Fura-2. Cells were paced at 0.5Hz with depolarising steps from -60mV to +10mV. Sarcoplasmic reticulum (SR) content was determined by application of caffeine (10-20mmol/l) and integration of inward NCX current. Rate constants for Ca2+ extrusion from the cell (kcaff) and Ca2+ uptake into the SR (kSERCA) were determined by fitting 1st order exponentials to decay phases of the respective Ca2+ transients5. Following an initial control protocol, a therapeutically relevant dose of Sil (1µM) applied. Data are presented as normalised to control for n cells. Differences between groups are determined with student’s paired t tests. Results: Sil reduced SR content by 26.5% (n=9, p<0.01). In addition, though to a lesser extent, Sil reduced Ca2+ transient amplitude (by -13.6%, n=9, p<0.05). This was not accompanied by a reduction in calculated SERCA activity (kSERCA -2.3% with Sil, p=0.97, n=5). Peak and integrated ICa,L were also reduced with Sil (-9.1% & -6.0%, respectively, n=9, p<0.05). This effect on ICa,L was also seen in adult dog ventricular myocytes (reducing peak and integrated ICa,L by 15.9% and 26.4% respectively, p<0.05 & p<0.01, n=6, temp 23C). Effect of Sil on integral ICa,L in ARVMs could not be ‘washed out’ by further application of control solution (washout -9.2%, p<0.05, n=8), and subsequent re-application of Sil reduced integral ICa,L even further (-15.2%, p<0.05, n=8). These effects cannot be attributed to ‘run-down’ effects. Conclusions: Sil substantially reduced SR content. Given that there was no significant reduction in kSERCA, this reduction may be mediated through ryanodine receptor modulation. Such reductions in SR content may reduce pro-arrhythmic SR Ca2+ release, and indicate a novel mechanism through which sildenafil exerts an antiarrhythmic effect. Acute reductions in Ca2+ transient amplitude and ICa,L with Sil indicate acute negative inotropic effects and may contribute to our understanding of its cardioprotective effects in the setting of hyperadrenergic drive in HF.