Herein we describe in vivo and in vitro assays to investigate the suspected antiandrogenic activity of two pyrethroids, cyfluthrin and beta-cyfluthrin. A stably transfected, androgen-responsive cell line, MDA-kb2, was used to determine the androgen receptor (AR) antagonistic effects of cyfluthrin and beta-cyfluthrin in vitro, and the Hershberger assay was utilized to detect the antiandrogenic potential of the two pyrethroids in vivo. Moreover, we also compared the antiandrogenic activities of cyfluthrin and beta-cyfluthrin to four structurally related pyrethoids: permethrin, cypermethrin, beta-cypermethrin and bifenthrin. Our results show that cyfluthrin and beta-cyfluthrin can block 5-dihydrotestosterone (DHT)-induced AR activity in MDA-kb2 cells. In the Hershberger assay, cyfluthrin, at doses of 18 and 54 mg/kg, and beta-cyfluthrin, at a dose of 36 mg/kg, caused significant decrease in the weight of seminal vesicle, ventral prostate, dorsolateral prostate, LABC, Cowper's glands, though not significant in glans penis. Beta-cyfluthrin at dose of 12 mg/kg decreased only the weight of seminal vesicle and had no effect on the other accessory sex tissues. The increase rank of antiandrogenic activity was: beta-cypermethrin<permethrin<beta-cyfluthrin<cypermethrin<cyfluthrin<bifenthrin<flutamide. In conclusion, cyfluthrin and beta-cyfluthrin are moderate antiandrogenic chemicals in our experiments, and they elicit antiandrogenic effects at least partly by antagonizing AR.