The aim of the present study was to investigate the effect of N-acetylcysteine (NAC) on freezability of Tushin ram semen. Ejaculates from four Tushin rams were collected with artificial vagina and then pooled. Pooled semen was divided into four aliquots to be diluted with skim milk-based-egg yolk-glycerol (SEG) extender supplemented with various concentrations of NAC (0, 0.25, 0.5 and 0.75 mM). The semen was loaded into 0.25 mL straws, equilibrated (at 4 degrees C for 2 h), frozen in liquid nitrogen (LN) vapour (at -120 degrees C for 15 min) and stored in LN (-196 degrees C). After thawing (at 37 degrees C for 1 min), sperm motility, dead-live ratio, morphology, abnormal acrosome and membrane integrity (HOST) were evaluated. Results showed that 0.75 mM NAC has detrimental effects on motility, compared to the other three NAC doses evaluated (P<0.05). Membrane integrity was higher in 0.25 and 0.5 mM NACs. There was significant differences in semen viability among NAC doses (P<0.05). In conclusion, higher doses of NAC, especially used with SEG extender, may have some detrimental effects on freezability of ram semen. Moreover, although modest doses of NAC slightly improved freezability of Tushin ram semen.