Ethylene glycol monoethyl ether (EGEE) is known to have testicular toxicity. To elucidate whether EGEE has any effect on sperm motion, especially in the case of short time exposure, we conducted a series of in vivo experiments with rats, as well as an in vitro study with rat sperm. Sperm from cauda epididymides and spermaducts was analyzed for the change in motion with a Hamilton-Thorne Sperm analyzer. Administration of EGEE at 600 mg/kg/d for five weeks significantly decreased total and progressive motility of sperm to 15-30% of controls, in both the cauda epididymis and the spermaduct. The time-course experiment using a single dose of 1,000 mg/kg showed that damage to sperm motion was evident at 12-24 h after EGEE administration. Addition of EGEE to the medium of sperm had no effect on its motion, but the metabolite ethoxyacetic acid showed a significant inhibitory effect. These results suggest that besides its toxicity to spermatogenesis, the metabolite of EGEE may also directly affect the motion of mature sperm.