Many toxic effects of treated wastewater effluent on organismal and reproductive health have been documented. However, the physicochemical environment of treated wastewater effluent frequently differs considerably from that of its receiving waters and may affect organismal function independently of toxic effects. Teleost sperm, for example, may be affected by the higher osmolality of treated wastewater, as this sperm is activated for a brief period of time following ejaculation due to the sudden decrease in osmolality of its surrounding environment. In this study, we examined the effects of treated wastewater effluent on sperm motility to test the hypothesis that the higher osmolality of effluent compared to river water will adversely affect sperm activation in a concentration-dependent relationship. Treated wastewater effluent was collected on 5 days from the outflow of the Metropolitan Wastewater Treatment Plant, St. Paul, Minnesota, and from an upstream site on the Mississippi River. Milt aliquots collected from goldfish were diluted in an isotonic extender solution and subsequently activated in either deionized water, 100%, 50%, or 10% effluent, a synthetic ion mixture, or river water. Sperm motility and velocity were assessed at 15-s intervals for 1 min using a computer assisted sperm analyzer. Significant differences in performance parameters were found only at 15 s, with sperm motility and velocity declining rapidly at later sampling times. Predictably, deionized water resulted in the greatest activation of sperm motility, while motility exhibited a concentration-dependent decline in 10%, 50%, and 100% treated wastewater effluent. Interestingly, Mississippi River water and a synthetic ion mixture with an osmolality comparable to 50% effluent both resulted in the least amount of sperm activation. However, sperm activation in river water varied between collection days during the study. River water and 100% effluent both had low sperm activation characteristics despite a 10-fold difference in osmolality between these two treatments (1 and 10 mOsmol kg(-1), respectively). Results of this study indicate a concentration-dependent decrease in sperm motility in treated wastewater effluent as well as significant fluctuations of sperm activation in Mississippi River water. This study illustrates the complexity of assessing the effects of treated wastewater effluents and the difficulty of determining appropriate reference sites for such studies.