['Sperm DNA fragmentation is a possible predictive parameter for male fertility status. The occurrence of M540 bodies in semen of subfertile subjects affects flow cytometric investigations in sperm. We set up a new method to evaluate DNA fragmentation excluding M540 bodies.', 'DNA fragmentation was evaluated by flow cytometry in semen of 75 subjects both by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL, traditional method) and by double staining with TUNEL and propidium iodide (PI, new method).', 'The use of the new method revealed that TUNEL underestimates sperm DNA fragmentation in flow cytometry and showed two sperm populations stained with low (PI(dim)) and high (PI(br)) avidity for PI. The PI(dim) population is entirely composed of DNA fragmented sperm and its incidence shows highly significant negative correlations with morphology, motility, sperm count and concentration (respectively, r = -0.51, -0.52, -0.46 and -0.32, n = 75). DNA fragmentation in the PI(br) sperm population is independent from semen quality.'] Does nuclear staining identify two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters?