Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein, which protects biomembranes from oxidative damage, and accounts for most of the selenium content in the mammalian testis. There are at least three different variants of this protein: cytosolic, mitochondrial and nuclear. In the rodent testis, PHGPx is associated with cytoplasm, mitochondria and nucleus of spermatogenic cells. In the late stages of spermatogenesis, reduction of hydrogen peroxide by PHGPx is coupled to protein sulfhydryl oxidation and formation of cross-links. Protein thiol oxidation catalyzed by PHGPx is functional for both the stabilization of spermatozoal structural protein and the chromatin condensation in the nucleus. However, its enzymatic activity is minimal in mature spermatozoa where PHGPx acquires a structural function, constituting the major component of the mitochondrial capsule Our objectives were to evaluate the protein activity in the spermatozoa of stallions of normal seminal characteristics and fertility. Three semen samples were collected by artificial vagina from six trotter stallions (n = 18) aged 12 to 22 years and actively breeding in a Standardbred studfarm located in Northeast Italy. After collection, each semen sample was filtered and volume, concentration of spermatozoa and motility were recorded. Part of the raw semen was diluted with phosphate-buffered saline (PBS) to a final concentration of 50×106 spermatozoa/ml and centrifuged (600×g) at room temperature for 15 min; the supernatant containing seminal plasma was aspirated and discharged and the sperm pellet was re-suspended with fresh PBS and centrifuged again at the same speed. The assessment of sperm integrity was checked by microscopy and the purified sperm samples were frozen at −20 ◦C for further analysis for PHGPx. Recovered enzymatic activity of PHGPx was determined from the time course of absorbance decrease at 340 nm (ε = 6.22mM−1 cm−1 for NADPH). Data are presented as means±S.D. Seminal characteristics of the experimental animals were in the normal range for the breed (ejaculate volume 41.6±18.7 ml, spermatozoa concentration ranged from 73 to 504×106 ml (189.8±119.9), and spermatozoa progressive motility ranged from 50 to 85% (74.8±12.4). The PHGPx activity was 185.3±95.9 (mU/mg protein). No relationships were found between seminal characteristics and PHGPx activity. Enzymatic activity was relatively constant within subject with two animals (stallions 1 and 2) having greater values compared to the other four. Further screening of a larger number of stallions and particularly of animals with impaired semen quality and fertility is necessary for a more complete understanding of the importance of this enzyme in equine spermatogenesis.