Spermiation, the process in which vertebrate spermatozoa are detached from investing Sertoli cells into the lumen of the seminiferous tubule, is a prerequisite for the successful fertilization. Using an in vitro Rana nigromaculata spermiation bioassay, we have shown that gonadotropin initiates spermiation by inducing the synthesis of delta 4-steroids by testis fragments. Among all of the delta 4-steroid metabolites produced by R. nigromaculata testis fragments, spermiation-inducing activity was confined to only one metabolite; this metabolite was identified as 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17 alpha,20 alpha-DP). Induction of spermiation by gonadotropin in vitro was accompanied by marked elevations in 17 alpha,20 alpha-DP concentrations in incubation media. These findings provide evidence that 17 alpha,20 alpha-DP is the nautrally occurring spermiation-inducing hormone in R. nigromaculata.