Background Acute intestinal toxicity is a frequent complication that may lead to interruption of treatment in patients undergoing pelvic radiotherapy. Reliable, non-invasive biological markers to evidence their severity are not yet available. Aim To test faecal DNA and calprotectin as potential biomarkers of intestinal toxicity caused by pelvic radiotherapy. Methods Patients were categorized according to the location of the cancer as nonrectal (n = 25) and rectal (n = 27). Four stool samples were collected at weeks w0, w3, w5 (end of radiotherapy) and w7. Faecal DNA was determined by quantitative PCR and calprotectin by ELISA. Intestinal toxicity was scored according to the Common Toxicity Criteria. Results In the nonrectal group, acute diarrhoea toxicity was present in 80% of patients, faecal DNA increased 10-fold during radiotherapy (1.5 × 103 copies/mg dry weight, 9.5 × 102-8.8 × 103 at w0, median and interquartile range vs. 1.3 × 104, 1.9 × 103-3.9 × 104 at w5, P < 0.01), but was not recovered at w7 (3.4 × 103, 1.5 × 103-4.1 × 104) and calprotectin doubled during treatment at w3 and w5. No significant changes in faecal markers were found in the rectal group. Conclusion Faecal excretion of human DNA and calprotectin increased during pelvic radiotherapy treatment, and may be a good objective biomarker of intestinal damage in nonrectal cancer patients. © 2009 Blackwell Publishing Ltd.