SUMMARY Sperm of the three-spined stickleback Gasterosteus aculeatus display a prolonged motility in the presence of ovarian fluid. The ovarian fluid prolongs sperm motility in freshwater from approximately 1 min to several hours, a trait that possibly gives the stickleback its unusual ability to spawn in waters of all salinities. The aim of the study was to look for factor(s) within the ovarian fluid responsible for prolonging sperm motility as well as to investigate the possible biological importance of the ovarian fluid under natural conditions. To that end, we measured the ionic content(Na + , Cl - , Ca 2+ and K + ) of the ovarian fluid and prepared ionic artificial ovarian fluids. We also prepared a mannitol solution with the same osmolality as the ovarian fluid in order to distinguish between the ionic and osmotic effect. We found that the ionic artificial fluids were equally effective as the natural ovarian fluid in prolonging sperm motility and survival over a range of concentrations, whereas the mannitol solution was far less effective. By insertion of natural ovarian fluid or ovarian fluid from which macromolecules had been removed by ultra filtration in nests it was found that macromolecules help by retaining ions. We also found that ovarian fluid in water, at concentrations as low as 0.75 and 1.56%, prolonged sperm motility and that the concentration of ions(Na + ) present in the nest 15 min after spawning corresponded to at least 3% ovarian fluid. Previous fertilisation experiments have shown that it takes at least 15 min for stickleback sperm to fertilise all eggs in a batch. This indicates that the role of ovarian fluid in prolonging the sperm motility is biologically relevant and that the effect is exerted by the fluid's ionic content.