Sperm were incubated for up to 9 days in the presence or absence of exogenous hydrogen peroxide, phenylalanine, catalase and aurintricarboxylic acid to assess the influence of reactive oxygen species and inhibition of deoxyribonucleases on sperm chromatin stability. The assessment of sperm DNA susceptibility to in situ acid denaturation by the sperm chromatin structure assay did not detect any difference in chromatin stability between sperm incubated for 9 days under aerobic and anaerobic conditions in a diluent called 14G. Exposure to exogenous hydrogen peroxide under both aerobic and anaerobic conditions and to phenylalanine under aerobic conditions (which produces hydrogen peroxide by a reaction catalysed by the aromatic amino acid oxidase present in sperm) was detrimental to sperm chromatin stability, increasing its DNA susceptibility to in situ acid denaturation over the incubation time. This effect was eliminated if catalase was present in the diluent. Inclusion of the general deoxyribonuclease inhibitor aurintricarboxylic acid in the diluent severely decreased sperm chromatin stability under both aerobic and anaerobic conditions. Aurintricarboxylic acid was mildly cytotoxic, as revealed by viability assessment, under aerobic, but not under anaerobic, incubation conditions. Exogenous hydrogen peroxide, either directly added to the diluent or generated through the enzymatic oxidation of phenylalanine, was detrimental to sperm motility and the integrity of the plasma membrane.