Inflammation activates a variety of inflammatory cells, which induce and activate several oxidant-generating enzymes which are able to produce high concentrations of free radicals and oxidants which react with each other to generate other more potent reactive oxygen and nitrogen species such as peroxynitrite (ONOO−) that can damage DNA, RNA, lipids, and proteins by nitration, oxidation, etc., leading to increased mutations and altered functions of enzymes and proteins. Spermatozoa generate small amounts of O2− and NO·. Under physiological condition these compounds exist at very low concentration; however their local concentrations become significant close the production sites and the formation of peroxynitrite appears likely. Moreover, tyrosine nitration is a widely used marker of peroxynitrite. The nitration of protein residues gives rise to 3-nitrotyrosine which represent a protein modification specific for ONOO− formation in vivo. In the present study we have determined ONOO− production in semen and its correlation with kinetic features in spermatozoa, and we set out to determine whether protein tyrosine nitration takes place in the same sample. Semen samples from 25 normal fertile donors (control group) and 40 infertile patients affected by idiopathic asthenozoospermia were analysed according to WHO 1999 criteria. After liquification one aliquot of semen diluted to 5×106 spermatozoa/ml was stored at −80 °C until determinations. Peroxynitrite concentration was measured through the fluorescence of the DCFDA probe. Protein tyrosine nitration was determined with Western Immuno Blot. Curvilinear velocity and straight line velocity of sperm cells were determined using a Motion Analysis CASA system. The controls exhibited ONOO− production significantly lower than asthenozoospermic patients (7.32±0.54 vs 27.16±1.58, P<0.001); furthermore, ONOO− exhibited a significant inverse correlation with the motility parameters. Moreover, in the western immuno blot there was an increase in the nitration of the tyrosine residues in the asthenozoospermic samples compared to controls. The present data suggest a critical negative effect of peroxynitrite on sperm motility when spermatozoa concentration is normal. Tyrosine nitration is enhanced by peroxynitrite that affects motility parameters. Thus, a possible pathogenic role in infertile men when asthenozoospermia is the main critical problem may be suggested.