Differential protein profiling by 2-D PAGE is generally useful in biomarker discovery, proteome analysis and routine sample preparation prior to analysis by MS. The goal of this study was to compare 2-D PAGE-resolved protein profile of lymphatic endothelial cells to those of venous, and arterial endothelial cells isolated from lymphatic and blood vessels of bovine mesentery (bm). Three 2-D PAGE electrophoretograms were produced for each of the three cell types and quantitatively analyzed. Protein identification by LC-MS/MS was performed to identify 39 proteins found to be present at statistically significantly different levels in the three cell types (p<0.05). Most of the 39 proteins have not been previously reported in EC proteomic studies of 2-D PAGE electrophoretograms. Three proteins, HSPA1B (HSP70 family member), HSPB1 (HSP27 family member), and UBE2D3 (a member of E2 ubiquitin-conjugating enzymes) found to be at highest levels in bm arterial endothelial cells, bm venous endothelial cells, and bm lymphatic endothelial cells, respectively, were validated by immunoblotting with appropriate antibodies. The lack of substantial overlap between our results and those of other groups' comparative studies are discussed. Functional implications of differences in levels of various proteins identified in the three cell types are also discussed.