The aim of this study was to investigate the activation of the p53 pathway and the induction of apoptosis during preoperative radiotherapy in normal human rectal tissue and in rectal carcinoma. Twelve patients with rectal cancer of the lower third were enrolled in this study. Tumor specimens and adjacent normal tissue were obtained before radiation, after the third radiation cycle and from the surgically removed rectum. All specimens were analyzed be means of immunohistochemistry for expression of p53 and its downstream target genes MDM2 and p21. In normal mucosal crypts, irradiation led to p53 accumulation and MDM2 induction in more than 70% of the cells. The accumulation of p53 in basal crypts was associated with high expression of p21. Apoptosis was also induced in crypts and occurred in 15% of the cells. Activation of the p53 pathway was not seen in the resting cells at the luminal border of the epithelium. In interstitial cells, p21 was highly upregulated, whereas p53 and MDM2 showed weak expression. The level of bcl-2 was not altered during radiotherapy in healthy tissue. In rectal carcinoma cells, p53 expression was unaltered by irradiation in 11 out of 12 tumors. The p53 non-functional tumors were characterized by a weak induction of MDM2 and p21 and by the lack of apoptosis in the presence of bcl-2. Our findings demonstrate that sequential immunohistochemical analysis is suitable to detect a deregulation of the p53 pathway in human rectal cancer cells during radiotherapy. Further investigations are necessary to elucidate its value as a prognostic marker and potential predictor of therapy responsiveness.