Signal transduction involves posttranslational modifications and protein-protein interactions, which can be studied by proteomics. In Arabidopsis, the steroid hormone (brassinosteroid, BR) binds to the extracellular domain of a receptor kinase (BRI1) to initiate a phosphorylation/dephosphorylation cascade that controls gene expression and plant growth. Here we detected early BR signaling events and identified early response proteins using prefractionation and two-dimensional difference gel electrophoresis (2-D DIGE). Proteomic changes induced rapidly by BR treatments were detected in phosphoprotein and plasma membrane (PM) fractions by 2-D DIGE, but not in total protein extracts. LC-MS/MS analysis of gel spots identified 19 BR-regulated PM proteins and 6 proteins from phosphoprotein fractions. These include the BAK1 receptor kinase and BZR1 transcription factor of the BR signaling pathway. Both proteins showed spot shifts consistent with BR-regulated phosphorylation. In addition, in vivo phosphorylation sites were identified for BZR1, two TPR proteins and a phosphoenolpyruvate carboxykinase (PCK1). Overexpression of a novel BR-induced PM protein (DREPP) partially suppresses the phenotypes of a BR deficient mutant, demonstrating its important function in BR responses. Our study demonstrates that prefractionation coupled with 2-D DIGE is a powerful approach for studying signal transduction.