Reactive oxygen species (superoxide anion, hydrogen peroxide, and nitric oxide) are involved in human sperm capacitation and associated tyrosine (Tyr) phosphorylation through a cAMP- and protein kinase A-mediated pathway. Recently, we evidenced the double phosphorylation of the threonine-glutamine-Tyr motif (P-Thr-Glu-Tyr-P) in human sperm proteins of 80 and 105 kDa during capacitation. The objective of the present study was to investigate the role of reactive oxygen species in the regulation of this process and to immunolocalize the P-Thr-Glu-Tyr-P motif in human spermatozoa. Superoxide dismutase and catalase did not prevent, and exogenous addition of superoxide anion or hydrogen peroxide did not trigger, the increase in P-Thr-Glu-Tyr-P related to sperm capacitation. However, l-NAME (a competitive inhibitor of l-arginine for nitric oxide synthase) prevented, and a nitric oxide donor promoted, the increase in P-Thr-Glu-Tyr-P related to sperm capacitation. In addition, l-arginine reversed the inhibitory effect of l-NAME on capacitation and the associated increase of P-Thr-Glu-Tyr-P. Therefore, the regulation of P-Thr-Glu-Tyr-P is specific to nitric oxide and not to superoxide anion or hydrogen peroxide. The nitric oxide-mediated increase of P-Thr-Glu-Tyr-P involved protein Tyr kinase, MEK or MEK-like kinase, and protein kinase C but not protein kinase A. The P-Thr-Glu-Tyr-P motif was immunolocalized to the principal piece region of spermatozoa. In conclusion, nitric oxide regulates the level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation. These data evidence, to our knowledge for the first time, a specific role for nitric oxide in signal transduction events leading to sperm capacitation.