The purpose of this study was to investigate the efficiency of the water test to evaluate the functional membrane integrity of ejaculated canine sperm after collecting, cooling, and freezing-thawing. Twenty-five ejaculates were obtained from 12 stud dogs by digital manipulation. Sperm-rich fractions were evaluated, diluted, cooled at 5ºC and frozen-thawed. A conventional hypo-osmotic swelling test (HOST) using a fructose solution (60 mOsm/l) was compared with a HOST using distilled water (0 mOsm/l), for evaluation of the functional integrity of the plasma membrane in fresh, cooled, and frozen-thawed semen. Distilled water detected a higher percentage of reacted sperm in fresh semen (91.2 ± 0.1%) than the conventional HOST (90.6 ± 0.1%; P < 0.05). For cooled and frozen-thawed semen, the water test was as efficient as the conventional HOST (81.2 ± 0.3% and 80.3 ± 0.3%, respectively), for detecting functional membrane integrity (P > 0.05). Regardless of the test used, there was a decrease in the mean percentage of reacted sperm after equilibrium and freezing-thawing (P < 0.05). In conclusion, this study demonstrated that water can efficiently replace conventional fructose-based hypo-osmotic media for evaluation of the functional integrity of the plasma membrane of ejaculated fresh, cooled, and frozen– thawed canine sperm.