This pilot study aimed to assess an original test based on the analysis of exfoliated colonocytes as a new approach to colorectal cancer (CRC) detection. DNA was isolated from exfoliated cells collected from the surface of the rectal mucosa by a standardized minimally invasive procedure in a case-control trial involving 66 patients with CRC diagnosis and 110 healthy volunteers (age 50-70). PicoGreen staining and quantitative real-time PCR (QRTPCR) were used for DNA quantification. Mean DNA scores in microg/ml obtained for the control and cancer groups were 2.1 (95% CI 1.7-2.5) and 9.0 (CI 6.7-11.2) respectively (p < 0.001) for PicoGreen and 0.8 (CI 0.6-0.9) and 3.8 (CI 1.9-5.7) respectively (p = 0.003) for QRTPCR. The PicoGreen assay better detected CRC presence. At DNA score cut-off point of 2.5 microg/ml this assay gave sensitivities of 77.8% (CI 52.4-93.6) for proximal tumours, 91.4% (CI 76.9-98.2) for distal CRC and 86.8% (CI 74.7-94.5) for all CRC with specificity at 74.0% (CI 64.0-82.4). Increasing the cut-off point to 5.0 microg/ml resulted in sensitivities of 38.9% (CI 17.3-64.3) for proximal tumours, 71.4% (CI 53.7-85.4) for distal CRC and 60.4% (CI 46.0-73.5) for all CRC. Specificity for this cut-off point increased to 94.8% (CI 88.3-98.3). The new procedure of exfoliated cell collection from the surface of the rectal mucosa is a simple, safe and well-tolerated technique providing high quality cells. These early results suggest that exfoliated cell collection in combination with DNA quantification can potentially be employed as a tool for CRC early detection.