OBJECTIVE Various protein contents such as enzymes, growth factors, and structural components are responsible for biological activities in organs. We have created a map of vitreous proteins and developed a proteome analysis of human vitreous samples to understand the underlying molecular mechanism and to provide clues to new therapeutic approaches in eyes with proliferative diabetic retinopathy (PDR). METHODS Vitreous and serum samples were obtained from subjects with idiopathic macular hole (MH, 26 cases) and PDR (33 cases). The expressed proteins in the samples were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Protein spots were visualized by silver staining, and their expression patterns were analyzed. Some protein spots of concern were excised from the 2-D gels, digested in situ with trypsin, and analyzed by mass spectrometry. RESULTS More than 400 spots were detected on 2-D gels of MH cases, of which 78 spots were successfully analyzed. The spots corresponded to peptide fragments of 18 proteins, including pigment epithelium-derived factor, prostaglandin-D2 synthase, and interphotoreceptor retinoid-binding protein. These were not identified in the corresponding serum samples. These proteins were also expressed in PDR samples, with no distinct tendency to increase or decrease compared with the MH samples. More than 600 spots were detected on 2-D gels of PDR cases, of which 141 spots were successfully analyzed. The spots corresponded to peptide fragments of 38 proteins. Enolase and catalase were identified among four detected spots. Neither was found in MH vitreous or in PDR serum samples. CONCLUSIONS A map of protein expression was made in human vitreous from eyes with MH and PDR. In the PDR eyes, the increased protein expression observed was due to barrier dysfunction and/or production in the eye. Proteome analysis was useful in systematic screening of various protein expression in human vitreous samples.