The renal proximal convoluted tubule is the primary site of water, electrolyte and nutrient reabsorption and of active secretion of selected molecules. Proteins in the apical brush-border membrane facilitate these functions and initiate some of the cellular responses to altered renal physiology. The current study uses two-dimensional liquid chromatography/mass spectrometry to compare brush border membrane vesicles isolated from rat renal cortex (BBMV(CTX)) and from purified proximal convoluted tubules (BBMV(PCT)). Both proteomic data and Western blot analysis indicate that the BBMV(CTX) contain apical membrane proteins from cortical cells other than the proximal tubule. This heterogeneity was greatly reduced in the BBMV(PCT). Proteomic analysis identified 193 proteins common to both samples, 21 proteins unique to BBMV(CTX), and 57 proteins unique to BBMV(PCT). Spectral counts were used to quantify relative differences in protein abundance. This analysis identified 42 and 50 proteins that are significantly enriched (p values <or=0.001) in the BBMV(CTX) and BBMV(PCT), respectively. These data were validated by measurement of gamma-glutamyltranspeptidase activity and by Western blot analysis. The combined results establish that BBMV(PCT) are primarily derived from the proximal convoluted tubule (S1 and S2 segments), whereas BBMV(CTX) include proteins from the proximal straight tubule (S3 segment). Analysis of functional annotations indicated that BBMV(PCT) are enriched in mitochondrial proteins and enzymes involved in glucose and organic acid metabolism. Thus the current study reports a detailed proteomic analysis of the brush-border membrane of the rat renal proximal convoluted tubule and provides a database for future hypothesis-driven research.