We have previously demonstrated the predominant localization of tyrosine-phosphorylated proteins in podocytes in the glomerulus of normal rat kidney by immunoblotting and immunohistochemical analyses using anti-phosphotyrosine antibodies. The five major phosphoproteins (70-, 80-, 120-, 145- and 220-kDa), which were detected on 1-DE immunoblotting analysis, were spitted into multiple spots on 2-DE gels. A newly developed technique for precise matching of immunoreactive spots to corresponding spots on silver-stained 2-DE gel, in combination with a comprehensive analysis of immuno-affinity purified tyrosine-phosphorylated proteins, successfully identified 8 proteins including novel phosphoproteins and 3 co-immunoprecipitated tyrosine kinases as shown in Table. Based on these findings we speculate that tyrosine-phosphorylation of membrane cytoskeleton associated proteins and other signaling molecules play roles not only in maintain the unique cytoskeleton organization in the podocyte but also in signal transduction of the glomerulus.