Contamination of canine semen with urine can drastically reduce sperm motility. To assess whether this effect is associated with differences in pH and osmolarity, the present study evaluated canine semen diluted in prostatic fraction, autologous urine, ultrapure water and NaCl solutions containing the following osmolarities: 133, 260, 392, 519 and 860 mOsmol/l. The semen dilutions were incubated for 1 h at 37°C. Total and progressive sperm motility, membrane and acrosomal integrity and sperm morphology were evaluated. Semen and urine pH values were similar (semen = 6.5 ± 0.3; urine = 6.9 ± 0.3). After 1 h of incubation, total and progressive motility in the prostatic fraction were similar to that of sperm in the 260 mOsmol/l solution (nearly 75% for total motility and 26-40% for progressive motility) and higher than observed in the other solutions, which had a total motility lower than 35% and a progressive motility under 3.2%. Compared to sperm in the seminal plasma, membrane integrity was lower in sperm incubated in water, solutions with 133 and 860 mOsmol/l and urine (0-50% integrity), and acrosomal integrity was lower in sperm incubated in water, 860 mOsmol/l solution and urine (0-32% integrity). Morphology changes (bent tail) were detected only in sperm incubated in the most hyposmotic solutions (water and 133 mOsmol/l). In conclusion, pH is most likely not associated with the deleterious effects of urine on semen quality, and although only the most hyposmotic solutions caused morphological changes in sperm, sperm motility is compromised in solutions that are highly hypo- or hyperosmotic, such as urine.