The effects of extracellular ice and cryoprotective agents on the measured volumetric shrinkage response and the membrane permeability parameters of equine spermatozoa have been reported previously. The volumetric shrinkage data were obtained using a differential scanning calorimeter technique that was independent of cell shape. The aim of this study was to examine the effects of collection and cooling conditions on the motility and the water transport parameters at subzero temperatures of equine spermatozoa. Stallion semen samples were collected using either a commercial lubricating agent, which caused osmotic stress to the spermatozoa, or water-insoluble Vaseline™ as the artificial vagina lubricant. In some experiments, spermatozoa were cooled at 1°C min-1 from 20°C to 4°C to induce cold shock. An Equitainer was used to achieve control cooling rates (≤ 0.3°C min-1) at temperatures > 0°C. The water transport response of spermatozoa that were cold-shocked and osmotically shocked was significantly different from that of control spermatozoa (P < 0.01). Osmotic stress appeared to have an effect on the water transport response, although this effect was not significant. These results indicate that cold shock alters the behaviour of equine spermatozoa in cryopreservation protocols as a result of changes in the water transport properties of the plasma membrane. Although osmotic stress did not significantly affect water transport in equine spermatozoa, it did significantly decrease sperm motility in the extended semen samples (P < 0.01), which would, in turn, lower the quality of cold-stored or cryopreserved spermatozoa.