PURPOSE: In the recent past, the potential suitability of fixed samples to 2-D DIGE studies has been demonstrated on model tissues, but not on "real-world" archival tissues. Therefore, this study was aimed to assess the quality of the results delivered by 2-D DIGE on samples retrieved from hospital tissue repositories. EXPERIMENTAL DESIGN: Diseased and normal tissue samples (namely, human gastric adenocarcinoma and normal gastric tissue, human lung neuroendocrine tumors, canine mammary tubulo-papillary carcinoma and normal mammary tissue, sheep liver with cloudy swelling degeneration and normal liver tissue) were retrieved from human and veterinary biorepositories and subjected to full-length protein extraction, cyanine labeling, 2-D DIGE separation, image analysis, MS analysis, and protein identification. RESULTS: Archival samples could be successfully subjected to 2-D DIGE, providing maps of satisfactory resolution, although with varying pattern complexity (possibly influenced by pre-analytical variables). Moreover, differentially expressed protein identities were consistent with the disease biology. CONCLUSIONS AND CLINICAL RELEVANCE: 2-D DIGE can support biomarker discovery and validation studies on large sample cohorts. In fact, although some information complexity is lost when compared to fresh-frozen tissues, their vast availability and the associated patient information can considerably boost studies suffering limited sample availability or involving long-distance exchange of samples.