The aim was to investigate the major C21 steroids produced by spermiating white croaker Micropogonias furnieri (Sciaenidae) in order to establish the potential mediator of gamete maturation in males of this species. The testes steroid production at the spawning season was identified incubating the H-3-17-hydroxy-4-pregnene-3,20-dione precursor through thin layer chromatography, high pressure liquid chromatography, enzymatic oxydation, acetylation and immunochemistry analyses. 17,20 beta-Dihydroxy-4-pregnen-3-one (17,20 beta-P) and 11 beta,17,21-Trihydroxy-4-pregnene-3,20-dione (cortisol) were the main metabolites produced. Contrary to what we expected, 17,20 beta,21-Trihydroxy-4-pregnen-3-one was not detected. Circulating levels of 17,20 beta-P were undetectable in immature testes and in those at the first spermatogenesis stages, while a clear increase was observed during the whole spermatogenesis and spermiation phases (from undetectable to 1047 pg mL(-1)). In vitro studies together with plasma detection suggest that 17,20 beta-P is a good steroid candidate involved in M. furnieri testes maturation. The role of cortisol during late phases of testes development needs further studies.