AbstractPHGPx of rat sperm mitochondrial capsule is cross‐linked and inactive. The enzyme is in part released in an active form by mercaptoethanol. Treatment with H2O2 of reduced and solubilised capsule proteins, in the absence of any added reductant, results in: i) H2O2 consumption which depends on the presence of both, PHGPx activity and protein thiols; ii) protein thiol oxidation with a stoichiometry of 2 equivalents of thiol per mole of hydroperoxide and, iii) PHGPx inactivation and cross‐linking. SDS‐PAGE analysis of monobromobimane‐labeled proteins, following incubation with H2O2, shows that the oxidation takes place in specific bands in the area of 20 kDa.It is concluded that the protein thiol peroxidase activity of PHGPx is responsible for cross‐linking proteins in the mammalian sperm capsule and accounts for the selenium dependency of spermatogenesis.