Phosphodiesterases (PDEs) play a key role in the control of cardiac contraction. PDE inhibitors are associated with cardiotoxicity, and increased knowledge of cardiomyocyte PDEs is required for improved pharmaceutical development. PDE activity and function were compared in animal and human cardiomyocytes in order to determine a suitable model for the human response, and to generate new insights into the role of cardiomyocyte PDEs. Ventricular myocytes were isolated from rat, guinea pig and diseased human heart. PDE gene expression was measured using RTPCR; PDE activity was measured using a scintillation proximity assay and isoform-specific PDE inhibitors. Effects of PDE inhibition on cardiomyocyte contractility were measured using electrically paced isolated cells. The human cardiomyocyte PDE expression and activity profile was closer to the guinea pig than the rat. High levels of PDE1 activity against cAMP and lower levels of possible PDE5 activity were found in guinea pig and human cardiomyocytes, but not rat. Non-specific PDE inhibition increased basal cardiomyocyte percentage shortening in cells from all three species; PDE3 inhibition only increased percentage shortening in guinea pig and human cardiomyocytes; PDE4 inhibition did not affect basal percentage shortening in cardiomyocytes from any of the three species. Further studies using rat cardiomyocytes found that PDE3 and PDE4 inhibition both significantly increased percentage shortening when cAMP levels were raised; rat β1- and β2- adrenergic receptors were also differentially regulated by PDE3 and PDE4. The functional effects of PDE1 inhibition were investigated in guinea pig cardiomyocytes but no significant effects were measured. Dual PDE1/PDE5 inhibition was shown to have a small negative effect on isoprenaline-stimulated contractility in rat, guinea pig and human cardiomyocytes. This study has revealed new insights into cardiomyocyte PDEs and demonstrated that the guinea pig may be a more suitable model for diseased human cardiomyocytes than the rat.