Regulation of protein kinase activities is crucial in both physiology and disease, but analysis is hampered by the multitude and complexity of kinase networks. We used novel peptide array chips containing 1152 known kinase substrate sequences to profile different kinase activities in renal lysates from homozygous Ren2 rats, a model characterized by hypertension and angiotensin II (AngII)-mediated renal fibrosis, compared to Sprague Dawley (SD) controls and Ren2 rats treated with an ACE inhibitor (ACEi). Five-wk old homozygous Ren2 rats were left untreated or treated with the ACEi ramipril (1 mg/kg/day) for 4 wks; age-matched SD rats served as controls (n=5, each). Peptide array chips were incubated with renal cortical lysates in the presence of radioactively labeled ATP. Radioactivity incorporated into the substrate motifs was measured to quantify kinase activity. A number of kinases with modulated activities, which might contribute to renal damage, were validated by Western Blot, immunoprecipitation and immunohistochemistry. Relevant kinases identified by the peptide array and confirmed using conventional techniques included p38 MAP kinase and PDGFR, which were increased in Ren2 and reversed by ACEi. Furthermore, insulin receptor signaling was reduced in Ren2 vs control rats, and G-protein-coupled receptor kinase (GRK) activity decreased in Ren2+ACEi vs untreated Ren2. Array-based profiling of tissue kinase activities in AngII-mediated renal damage provides a powerful tool in identification of relevant kinase pathways in vivo and may lead to novel strategies for therapy.