Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2′-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p