The aim of the present study was to measure free thiols content, Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities in human spermatozoa of asthenozoospermic patients and normospermic donors, and evaluate any influence on kinetic sperm features, as well as correlation with peroxynitrite. In fact, membrane integrity and its composition are the basic characteristics of the sperm membrane; thus, it is evident that its composition is an important factor for membrane functions that can be modified upon oxidation. A total of 70 infertile patients affected by idiopathic asthenozoospermia and 25 normal fertile donors were enrolled. Control spermatozoa exhibited Na(+)/K(+)-ATPase, and Ca(2+)-ATPase activities, cytoplasmic Ca(2+) concentration and free -SH content significantly higher than those of asthenozoospermic patients (P < 0.0001). Moreover, positive associations were found between Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities and total sperm motility and sperm kinetic features, whereas negative associations were found between peroxynitrite and Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities, and total SH content. Peroxynitrite is able to reduce Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities and intracellular Ca(2+) concentration, through possible depletion of free thiols content. Decrease in motility and loss of sperm function in idiopathic asthenozoospermia can be attributed to these sulphydryl groups, important entities of the sperm membrane.