G protein-coupled receptor kinase-2 and -3 (GRK2 and GRK3)¹ in cardiac myocytes catalyze phosphorylation and desensitization of different G protein-coupled receptors through specificity controlled by their carboxyl-terminal PH domain. Whereas GRK2 has been extensively investigated, the function of cardiac GRK3 remains unknown. Thus, in the present study cardiac function of GRK3 was investigated in transgenic (Tg) mice with cardiac-restricted expression of a competitive inhibitor of GRK3, i.e. the carboxyl-terminal plasma membrane targeting domain of GRK3 (GRK3ct). Cardiac myocytes from Tg-GRK3ct mice displayed significantly enhanced agonist-stimulated ₁-adrenergic receptor-mediated activation of ERK1/2 versus cardiac myocytes from nontransgenic littermate control (NLC) mice consistent with inhibition of GRK3. Tg-GRK3ct mice did not display alterations of cardiac mass or LV dimensions compared with NLC mice. Tail-cuff plethysmography of 3 and 9 months old mice revealed elevated systolic blood pressure in Tg-GRK3ct mice vs. control mice (3 months old mice: 136.8±3.6 vs. 118.3±4.7 mmHg, p<0.001), an observation confirmed by radiotelemetric recording of blood pressure of conscious, unrestrained mice. Simultaneous recording of LV pressure and volume in vivo by miniaturized conductance-micromanometry revealed increased systolic performance with significantly higher stroke volume and stroke work in Tg-GRK3ct mice than in NLC mice. This phenotype was corroborated in electrically paced ex vivo perfused working hearts. However, analysis of left ventricular function ex vivo as function of increasing filling pressure disclosed significantly reduced (dP/dt)_min and prolonged time constant of relaxation (tau) in Tg-GRK3ct hearts at elevated, supraphysiological filling pressure compared with control hearts. Thus, inhibition of GRK3 apparently reduces tolerance to elevation of preload. In conclusion, inhibition of cardiac GRK3 causes hypertension due to hyperkinetic myocardium and increased cardiac output relying at least partially on cardiac myocyte ₁-adrenergic receptor hyperresponsiveness. The reduced tolerance to elevation of preload may cause impaired ability to withstand pathophysiological mechanisms of heart failure.