Nonylphenol (NP) is an important environmental toxicant and potential endocrine disrupting chemical. The objective of these studies was to determine the effects of NP on epididymal rat sperm in vitro. Epididymal sperm samples from Sprague-Dawley rats were incubated in 1, 10, 100, 250, and 500 microg/ml NP for 1, 2, 3, or 4h. Computer-assisted sperm analysis was used to determine motility. Epifluorescent microscopy was used to determine acrosomal status and flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity. Exposure of epididymal rat sperm to 250 or 500 microg/ml NP was highly detrimental to motility (P<0.05), with complete loss of motility observed after exposure to 500 microg/ml NP (P<0.05). The acrosomal integrity of sperm was significantly reduced with the lowest concentration (1 microg/ml) of NP, and higher concentrations resulted in a dose-dependent induction of the acrosomal reaction (P<0.05). Similarly, the percentage of sperm with high MMP declined dramatically after exposure to 100, 250, and 500 microg/ml NP (P<0.05). Duration of NP exposure did not have any effect on motility or MMP and NP did not appear to have detrimental effects on chromatin integrity (P>0.05). These results indicate that major mechanism of action of NP on rat sperm is by adversely affecting their acrosomal integrity. However, NP-induced impaired sperm motility, decreased mitochondrial membrane potential also likely to play an important role in destruction of sperm function.