The aim of the study was to develop a hypo-osmotic swelling test (HOS-test) for evaluating plasma membrane integrity in donkey spermatozoa. In the first study, six different hypo-osmotic solutions (fructose or fructose/sodium citrate, 75 or 150 mOsm, or bi-distilled water at 1 : 10 semen : solution ratio, or bi-distilled water at 1 : 3) were compared. The 75 mOsm fructose solution (1 : 10) and bi-distilled water (1 : 3) were chosen for study 2, where two incubation times (5 or 45 min) were tested. Bi-distilled water showed a significantly higher proportion of plasma membrane intact spermatozoa than the fructose solution (p < 0.05), it was thus concluded that the simple incubation for 5 or 45 min at 37 degrees C of one part of semen with three parts of bi-distilled water is an applicable HOS-test in the semen analysis of donkey spermatozoa. Regression analyses showed a significant correspondence of the latest method with Sybr 14- propidium iodide staining.