Despite the prevalence of zebrafish as a model scientific organism, understanding sperm function in this species is essentially limited to observations that osmotic shock initiates motility. During natural spawning, sperm encounter a range of environmental salinities as well as freshwater mixed with egg-associated ovarian fluid (OF), thus sperm are likely to be exposed to saline prior to egg contact. Effects of saline on sperm function in this model species are unknown, but likely to be important. Using computer assisted sperm analysis, this study addressed the effects of osmolality of spawning media and ionic composition and pH on the proportion of sperm becoming motile at activation (motility), as well as sperm velocity and path. When activated with tap water, motility was maximal (80%) at 10s (earliest time measured), declining to 5% by 87 s postactivation. With activation at moderate osmolalities ( approximately 160-200 mmol/kg) initial motility was decreased relative to low osmolality, increased from 10 to 30s, and subsequently declined less rapidly (motility in 80 mM NaCl was 35%, 80%, and 60% at 10, 30 and 147 s, respectively). Thus, moderate osmolality increased duration, but introduced a temporal lag in motility onset. With moderate osmolalities, the rate of velocity decay was less than that with tap water activation. Sodium chloride and sucrose similarly impacted both motility and velocity. Replacement of NaCl with KCl, pH values ranging from 6.8 to 8.4, or the presence of gadolinium were without effect. Motility, but not velocity, was slightly supressed by Ca(2+). Therefore, whereas pH and concentrations of Ca(2+) or K(+) of OF are unlikely to impact fertility via sperm motility, the OF contribution to spawning media osmolality may have pronounced effects on motility and velocity of sperm, factors previously correlated with fertility in other species.