G protein–coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including &agr;1B-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the &bgr;-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type &agr;1BAR. Transgenic mice with cardiac CAM&agr;1BAR overexpression had enhanced myocardial &agr;1AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAM&agr;1BARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAM&agr;1BAR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type &agr;1BAR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type &agr;1BAR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial &agr;1AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo &agr;1AR mitogen–activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with &agr;1BARs in vivo such that GRK3 desensitizes all &agr;1BAR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo &agr;1BAR signaling in the heart.