We examined the effects of maturation stimulating hormone, salmon pituitary extract (SPE) or 17α-hydroxyprogesterone (17α-OHP), injections on the post-thaw motility of ayu spermatozoa. Comparative post-thaw motility (percent motility of post-thawed spermatozoa/that of fresh spermatozoa x100) in the Ringer control group showed higher percent motility in the late time than in the early time of the spawning season. Post-thaw motility was higher in the SPE and 17α-OHP groups than in the Ringer control group. These results indicate that maturation of spermatozoa enhances the tolerance of ayu spermatozoa to cryopreservation.