This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze-thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5. mM), carnitine (2.5 and 7.5. mM), inositol (2.5 and 7.5. mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25. ml straws. Frozen straws were then thawed individually at 37 °C for 20. s in a water bath for the evaluation. The extender supplemented with 7.5. mM doses of carnitine and inositol led to higher subjective motility percentages (61.9 ± 1.3% and 51.3 ± 1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5. mM and inositol at doses of 7.5. mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5. mM ( P< 0.001). As regards CASA motility, 7.5. mM carnitine (41.6 ± 2.9% and 54.2 ± 4.9%) and inositol (34.9 ± 2.0% and 47.3 ± 2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5. mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage ( P< 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group ( P> 0.05). The maintenance of AOP level in methionine 2.5. mM was demonstrated to be higher (5.06 ± 0.38. mM) than that of control (0.96 ± 0.29. mM) following the freeze-thawing ( P< 0.001). Supplementation with these antioxidants prior to the cryopreservation process protected the DNA integrity against the cryodamage. Furthermore, future research should focus on the molecular mechanisms of the antioxidative effects of the antioxidants methionine, carnitine and inositol during cryopreservation. © 2010 Elsevier Inc.