Nonylphenol is one of the compounds believed to cause endocrine disruption and affect sperm quality in mammals. However, there is little information on its effects on sperm motility in fish or other forms of wildlife. We examined the effects of nonylphenol on the motility of spermatozoa of medaka (Oryzias latipes) using an in vitro bioassay. Sperm was activated in aqueous media containing no nonylphenol (solvent control, 0.1% ethanol) or 1 or 100 micromol/L nonylphenol and immediately loaded into a sperm motility observation chamber. The ratio of motile spermatozoa and their swimming speeds were analyzed by computer-assisted image analysis at 30 and 60 s after activation. A decrease in swimming speed or the ratio of motile spermatozoa was observed in spermatozoa exposed for 60 s to 100 micromol/L nonylphenol. Our results suggest that nonylphenol causes a reduction in sperm viability in teleost fish in the short period between ejaculation and fertilization.