The presence of androgen receptors in the ovaries of several vertebrate species, including Atlantic croaker, suggests that androgens may have important roles in ovarian function. In the current study the effects of androgens on ovarian steroidogenesis in Atlantic croaker were investigated. Addition of 17β-hydroxy-5α-androstan-3-one (DHT), 11-ketotestosterone (11-KT), or Mibolerone to ovarian incubations caused dose-dependent decreases in gonadotropin-stimulated in vitro estradiol production, which was not reversed by cotreatment with the antiandrogens, cyproterone acetate or 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene. Androgen treatment also caused significant decreases in estradiol production in the presence of 17-hydroxyprogesterone, which suggests that the site of androgen action is downstream of this steroid in the steroidogenic pathway. The mechanism of androgen action on ovarian steroidogenesis was also investigated. Coincubation with actinomycin D did not reverse the inhibitory effect of the androgens, which suggests that the mechanism of androgen action is nongenomic. An androgen conjugated to bovine serum albumin (DHT-BSA), which does not enter the cell, also caused inhibition of estradiol production in vitro, indicating that the androgen is acting at the cell surface. In addition, time course experiments revealed that the androgen action is rapid; 5-min exposure to DHT was sufficient to cause a significant reduction in estradiol production. Finally, preliminary evidence was obtained for the existence of a high-affinity, low-capacity androgen binding site in croaker ovarian plasma membranes. These studies suggest that androgens can down-regulate estrogen production in croaker ovaries via a rapid, cell surface-mediated, nongenomic mechanism.