The aim of this study was to evaluate in vitro motility and viability parameters when diluting the 5% post-thaw cryoprotectant dimethylformamide to concentrations of 2.5 and 1.25%, using two commercial diluents added to cryopreserved equine semen. After thawing, the samples were diluted in order to maintain the final concentrations (2.5 and 1.25%) of cryoprotectant, using two commercial diluents (FR4® and Botu-Crio®) at two times: initial (Ti) and final (Tf). Thirteen different ejaculate samples from five national breed stallions were used. Motility parameters were observed using computerized analysis and plasma membrane integrity using epifluorescence microscopy. There was an improvement in total and progressive sperm motility parameters at the final time (P<0.05) with Botu-Crio® diluent compared to FR4®. There was no difference, P>0.05, between the treatments in terms of plasma membrane integrity.