Posttranslational modification of target substrates underlies biological processes through activation/inactivation of signaling cascades. To concurrently identify the phosphoprotein substrates associated with cardiac β-adrenergic signaling, the mouse myocyte phosphoproteome was analyzed using 2-D gel electrophoresis in combination with ³²P autoradiography. Phosphoprotein spots, detected by silver staining, were identified using MALDI-TOF mass spectrometry in conjunction with computer-assisted protein spot matching. Stimulation with isoproterenol (1 μmol/L for 5 minutes) was associated with maximal increases in myocyte contractile parameters, and significant stimulation of the phosphorylation of troponin I (190±23%) and succinyl CoA synthetase (160±16%), whereas the phosphorylation of pyruvate dehydrogenase (48±10%), NADH-ubiquinone oxidoreductase (46±6%), heat shock protein 27 (18±3%), αB-crystallin (20±3%), and an unidentified 26-kDa protein (29±7%) was significantly decreased, compared with unstimulated cells (100%). After sustained (30 minutes) stimulation with isoproterenol, only the alterations in the phosphorylation levels of troponin I and NADH-ubiquinone oxidoreductase were maintained and de novo phosphorylation of a phosphoprotein (≈20 kDa and pI 5.5) was observed. The tryptic peptide fragments of this phosphoprotein were sequenced using postsource decay mass spectrometry, and the protein was subsequently cloned and designated as p20, based on its high sequence homology with rat and human skeletal p20. The mouse cardiac p20 contains the conserved domain sequences for heat shock proteins, and the RRAS consensus sequence for cAMP-PKA substrates. LC-MS/MS phosphorylation mapping confirmed phosphorylation of Ser¹⁶ in p20 on β-agonist stimulation. Adenoviral gene transfer of p20 was associated with significant increases in contractility and Ca transient peak in adult rat cardiomyocytes, suggesting an important role of p20 in cardiac function. These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins to dynamically fine-tune cardiac responses to β-adrenergic signaling.