The introduction of preoperative radiotherapy (RT) in the treatment of rectal cancer has reduced the frequency of local recurrence and improved patient survival, and RT is now a part of the standard treatment regime in Sweden.However, it is still a major problem that there are unknown factors contributing to variations in recurrence and survival after RT and surgery among rectal cancer patients with the same turnour stage, therefore it is important to search for biological markers that might influence recurrence and survival, and to identify the patients who benefit from RT.The study included primary tumours from 163 rectal cancer patients who participated in a clinical trial of preoperative RT (87 patients without and 76 with RT before surgery), alone with the corresponding distant and adjuvant normal rectal mucosa, as well as lymph node metastasis.We have examined p53, p73, survivin, Cox-2, legumain, FXYD-3, MAC30, PRL, ATM, Ki-67, CD163 and apoptosis, by PCR using confronting two-pair primers and electrophoresis, immunohistochemistry, Western Blot and TUNEL.Expression of p73, Cox-2, legurnain, FXYD-3, MAC30, PRL aud ATM increased from either distant or adjacent normal mucosa to primary tumour.In the RT group, overexpression of p53, p73, Cox-2, legumain, FXYD-3 and PRL was related to less tumour necrosis or apoptosis, increased incidence of local or distant metastasis, and an unfavourable prognosis independent of both the tumour stage and differentiation.However, none of these effects was seen in the non-RT group.In further interaction analyses, the correlations with prognostic significance of these factors were different between the patients with RT and the patients without RT.In conclusion, certain biomarkers were independent prognostic factors in patients receiving preoperative RT for rectal cancer, which might provide additional information for selecting patients for preoperative RT.