Although hypoosmotic tests are widely used to assess spermatozoal quality in different species, they have not been used extensively in the stallion. Moreover, the role of the Na+/K+, ouabain sensitive-ATP-ase in the response of equine sperm to hypoosmotic shock is not well understood. This study tests two hypotheses: 1) that equine spermatozoa will respond to a hypoosmotic medium by swelling of the tail, and 2) that addition of ouabain will increase the percentage of swollen sperm tails. Ejaculates from 3 stallions were collected with an artificial vagina and diluted in Kenney's medium (Time = 0). Aliquots's were randomly selected to be incubated in an isoosmotic (297 mOsm) or different hypoosmotic media that were composed of citrate or of citrate with fructose. The osmolarity of the hypoosmotic media with citrate range from 18 to 96 mOsm, and the medium composed of citrate plus fructose (HOS medium) was of 153 mOsm. Moreover, aliquots of spermatozoa pretreated with ouabain were added to the isoosmotic medium an also to the HOS and the 96 mOsm citrate medium (ORT medium). Incubation of equine sperm in the hypoosmotic of equine sperm in the hypoosmotic media resulted in a time- and osmolarity-dependent swelling of the sperm tail, reaching maximum values after maximum values after incubation for 20-30 min in both the HOS and ORT media. Ouabain induced a dose-dependent effect on swollen tails and viability in fresh semen and also affected some parameters related to motility. Ouabain also increased the swelling response in a hypoosmotic medium although viability decreased. The percentage of swollen tails after incubation in ORT and HOS media showed significant correlations to viability, altered acrosomes and total motility, but not to other parameters of horse semen analysis. Our results suggest that hypoosmotic tests could be used to improve standard horse semen analysis. Additionally, Na+/K+-ATP-ase activity could be related to the response against hypoosmotic shock of horse spermatozoa.