The aim of this study was to verify the effect on equine sperm viability post-thawing by adding the cryoprotectant dimethyl formamide (5%) at 22°C, using a single step or by adding one tenth of the cryoprotectant at 1-min intervals into the extender, with or without slow cooling to 5°C before freezing in liquid nitrogen vapor. INRA 82 was used as the semen extender. Post-thawing sperm cells showed increased viability parameters (P < 0.05) when the cryoprotectant was added in one-tenth fractions, at 1-min intervals and using slow cooling to 5°C before freezing (P < 0.05). A second aim was to verify whether increasing the time interval between the addition of each tenth part of dimethyl formamide, cooling the samples to 5°C, increasing equilibration time and using different freezing rates would improve post-thawing sperm viability. Sperm viability analyses in this part of the study showed no significant improvement in sperm parameters in any of the treatments (P > 0.05). It may be concluded that the stepwise addition of dimethyl formamide at 1-min intervals to the INRA 82 extender and using slow cooling to 5°C before freezing, independent of the freezing system, preserved equine sperm viability post-thawing more than any of the methods tested.