Spermatozoa of marine teleosts, puffers and flounder, were completely quiescent when they were washed to remove electrolytic components of the seminal plasma and then diluted in nonelectrolyte solutions isotonic to the seminal plasma. Sperm motility was initiated upon dilution in hypertonic nonelectrolyte solutions. These observations suggest that sperm motility is suppressed by seminal osmolality and motility is triggered solely by the increase in external osmolality which occurs at natural spawning in hypertonic seawater. Extracellular Ca2+ had no influence on the osmolality-dependent initiation of sperm motility. However, sperm motility was initiated even in isotonic solution when Ca2+ was introduced into the sperm cells by Ca2+ ionophore. Intracellular Ca2+ increased at the osmolality-dependent initiation of sperm motility under Ca(2+)-free conditions. These results suggest that the release of Ca2+ from intracellular storage in response to the increase in external osmolality has a key role in the initiation of sperm motility. A transient increase in intracellular pH was also observed at the hyperosmolality-dependent initiation of sperm motility. Furthermore, initiation of sperm motility was induced even in isotonic solutions when intracellular pH increased by the treatment with ammonium salts. These results suggest that an increase in intracellular pH, as well as the rise in intracellular Ca2+, has an important role in the initiation of sperm motility in marine teleosts.