Background: Identification of signal transduction pathways that are critically involved in Alzheimer's disease (AD) is essential for the development of disease-specific biomarkers and drug therapy. Objective: This study is aimed at identifying protein kinases and signaling pathways that are activated in AD pathology. Methods: Microarray-based kinome profiling was employed for the detection of protein kinase activity in postmortem brain tissue derived from AD and age-matched nondemented control cases. Global serine/threonine kinase activity profiles are identified applying a peptide array system consisting of 140 peptides derived from known kinase substrate sequences covalently attached to porous chips, through which a protein solution is constantly pumped up and down. Peptide phosphorylation is determined by measuring the association of a mixture of fluorescently labeled antibodies, raised against phosphoserine- or phosphothreonine-containing peptides. Results: Protein lysates from freshly frozen postmortem brain tissue from nondemented controls and pathologically confirmed AD cases show ATP-dependent phosphorylation of peptides. In AD and control cases, peptides that are differentially phosphorylated are identified. Conclusion: Protein kinase activity profiling can be used to reveal novel kinases and new signaling pathways involved in AD pathology. Copyright © 2012 S. Karger AG, Basel.