Objective: The present study was designed to address the question of the contribution of cardiac troponin I (cTnI) phosphorylation to the enhanced rate of relaxation during β-adrenergic stimulation in hearts in situ. Methods: In situ hemodynamic measurements were performed in mouse hearts that (1) express normal level of phospholamban (PLB) and either express cTnI (PLB/cTnI) or the slow skeletal isoform of TnI (PLB/ssTnI) that cannot be phosphorylated by protein kinase A (PKA) or (2) do not express PLB and either express cTnI (PLBKO/cTnI) or ssTnI (PLBKO/ssTnI). Results: In the basal state, there was no difference in heart rate (HR), developed pressure (DP), left ventricular end-diastolic pressure (LVEDP) or rate of contraction (+dP/dt) between PLB/cTnI and PLB/ssTnI groups. However, hearts expressing ssTnI (PLB/ssTnI) showed a significantly decreased rate of relaxation (−dP/dt) when compared with hearts expressing cTnI (PLB/cTnI). In response to β-adrenergic agonist, isoproterenol (ISO), HR increased similarly in both groups. At the two highest doses of ISO, the rate of relaxation (−dP/dt) was significantly smaller in PLB/ssTnI than in PLB/cTnI hearts. In the basal state, there was no difference in HR, DP, LVEDP,+dP/dt and –dP/dt between PLBKO/cTnI and PLBKO/ssTnI hearts. In response to ISO, HR increased similarly in both groups and was only slightly smaller in PLBKO/ssTnI group at the lowest dose of ISO. However, during ISO perfusion, when cTnI was phosphorylated, the rate of relaxation was significantly slower in PLBKO/ssTnI compared to PLBKO/cTnI hearts. Conclusion: Our data support the hypothesis that phosphorylation of cTnI significantly contributes to the enhanced rate of relaxation during β-adrenergic stimulation.