Protein S-nitrosation represents a recently described form of post-translational modification that is rapid and reversible. However, the analysis of protein S-nitrosation in situ has been difficult because of the absence of specific probes and the instability of cellular protein S-nitrosothiols. We developed a rapid and specific method for detecting endothelial S-nitrosoproteins patterned after the biotin switch method that involves thiol alkylation followed by reductive generation of thiols from S-nitrosothiols, which are then labeled with either a biotin- or Texas red-derivative of methanethiosulfonate. When we used this methodology, we found that S-nitrosated proteins can form within endothelial cells from an exogenous S-nitrosothiol donor or from endogenous production of NO by endothelial NO synthase. When we used confocal microscopy, we found that these S-nitrosoproteins exist mainly in the mitochondria and peri-mitochondrial compartment, and that their half-life is approximately 1 h. Cellular S-nitrosated protein abundance changed as expected, with changes in activity of NO synthase, and with impairment of mitochondrial function and scavenging of peroxynitrite. We used a proteomic approach involving two-dimensional gel electrophoresis and mass spectrometry, and found that a limited number of S-nitrosoproteins exist in endothelial cells (S-nitrosoproteome) and identified GAPDH, vimentin, beta-galactosidase, peroxiredoxin 1, beta-actin, and ubiquitin-conjugating enzyme E2 among them. The most abundant S-nitrosated protein in the resting endothelial cell is GAPDH, suggesting a regulatory function for NO in glycolysis. These data offer methods and insights into identifying the protein targets of S-nitrosation reactions and their potential role in cell function and phenotype.