Radiation therapy (RT), combined with chemotherapy, is currently the standard adjuvant approach for UICC-stage II and III rectal cancer patients. Individual rectal tumors display wide ranges of radiosensitivity (RS). The aim of the present study was to identify proteins associated with radioresistance (RR), with the final aim of predicting tumor response. Seventeen patients were recruited between July 1998 and November 2001. All patients suffered from a locally advanced adenocarcinoma of the rectum. Tumor biopsies were taken before RT. All patients received preoperatively 50 Gray or a biologically equivalent total dose. Surgery was performed after 6 weeks, and response assessed histopathologically. Seven tumors showed complete response, seven a partial response, and in three patients only microscopic disease remained. Proteins were separated using narrow pH gradient two-dimensional polyacrylamide gel electrophoresis and silver staining. Automatic gel comparison allowed matching a mean number of 497 ± 280 spots. Forty-four spots showing significant differential expression in RR tumors were localized on the pH 4.5–5.5 gels, 33 out of them being identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Twenty-two out of 37 spots of interest could be identified on the pH 5.5–6.7 gels. The expression of the following proteins correlates with RR: tropomodulin (p = 0.01), heat shock protein 42 (p = 0.03), beta-tubulin (p = 0.10), annexin V (p = 0.10), calsenilin (p = 0.10), or with radiosensitivity (RS): keratin type I (p = 0.03), notch 2 protein homolog (p = 0.05) and DNA repair protein RAD51L3 (p = 0.11). A further RR-related protein is so far only hypothetical: XP_030188 (NCBI Database accession number, p = 0.14). We consider the fact that several of these proteins of interest are known to be associated with RR as a valid proof of principle for this proteomics approach. These results will serve as a basis for developing an assay for testing rectal cancers for radioresistance.