The short-term effects of estrogens and xenoestrogens on testicular androgen production were investigated in an in vitro incubation bioassay system using testicular tissue from the Atlantic croaker (Micropogonias undulatus). Incubation of testicular tissue fragments with estradiol over the concentration range of 37 nM to 37 μM caused concentration-dependent decreases in gonadotropin-stimulated 11-ketotestosterone (11-KT) production. The effect was specific for estrogens; progesterone, cortisol, and the synthetic androgen mibolerone did not significantly alter 11-KT production at similar concentrations. Diethylstilbestrol, the antiestrogen ICI 182,780, and several xenoestrogens including Kepone (chlordecone), 4-nonylphenol, and a hydroxylated polychlorinated biphenyl metabolite also significantly decreased gonadotropin-stimulated 11-KT production. The action of estradiol was rapid (<5 min) and was not blocked by actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively. Moreover, estradiol conjugated to BSA, which cannot pass through the cell membrane, also caused a decrease in 11-KT production. In addition, an estrogen-binding moiety was identified in testicular membrane preparations that had a single class of high-affinity (K_d 1.6 nM), saturable (1.2 nM), displaceable, finite (B_max 0.03 nM, 26 fmol/g testis) binding sites specific for estrogens and exhibited rapid association (t_1/2 = 5 min), characteristics typical of steroid membrane receptors. Overall the relative binding affinities of estrogens, other steroids, antiestrogens, and xenoestrogens for the membrane preparation correlated with their activities in the androgen production bioassay, thereby satisfying the final criteria for the designation of this estrogen-binding moiety as a steroid membrane receptor. The results demonstrate that estrogens and also probably xenoestrogens can act on the cell surface via a nongenomic mechanism to alter testicular androgen production in this vertebrate species.