The occurrence of DNA breaks in human sperm is of concern to genetic safety in artificial reproduction techniques. Here, we have explored the neutral comet assay (NCA) for evaluating the frequency of spermatozoa with double strand (ds) DNA breaks in normospermic donors. The NCA results into DNA tail formation by fibre extension and by the separation of DNA fragments. Gamma-irradiated native, lysed and lysed plus RNA and protein degraded human sperm nuclei have been used to assess sensitivity and specificity of fragment formation as an indication for ds DNA breaks. At 5 and 10 Gy gamma irradiation, the sensitivity increases in the order: native, lysed, lysed plus RNA and protein degraded. At 10 Gy, a uniform response between donors was obtained. For technical and biological reasons, the NCA underestimates the true incidence of ds DNA breaks by an unknown factor. Semen samples of six healthy normospermic donors were differentiated by swim up and by Percoll density centrifugation, followed by the NCA. In native semen, percentages of sperm nuclei with ds DNA breaks ranged from 15 to 25%. Swim up and selection for high-density sperm nuclei (high Percoll fraction) reduced the frequency of sperm with ds DNA breaks by about one third, whereas an increased frequency was found in the low Percoll fraction. In conclusion, the response to gamma irradiation of DNA fragment formation indicates the NCA to demonstrate ds DNA breaks which is in keeping with theory and experimental results from somatic cells. Ds DNA breaks are a characteristic of the sperm population of normal donors. Current sperm selection procedures reduce the fractions of sperm with ds DNA breaks, yet are not effective in eliminating these cells.