Abstract: The current study investiaged effects of 17-estradiol (E2), 17-methyl testosterone (MT) and androgen analogues androstendione (AED) exposure on brain tissue CYP19b gene expression in Gambusia affinis. The G. affinis CYP19b cDNA of full sequence was cloned and analyzed for the first time. G. affinis were randomly divided into five groups, with one control group and four experimental groups (2, 10, 50 and 100 ng/L E2, MT and AED, respectively). The CYP19b mRNA expression levels in fish brain tissues were determined after 1, 3, 5, and 7d exposure, respectively. The G. affinis CYP19b cDNA of full sequence was cloned, from which 1670 bp nucleotides were obtained, encoding 501 amino acids with an open reading frame (ORF) from 52 bp to 1554 bp. The comparison among the similarity amino acid sequence of CYP19b with other related fishes supports high similarity between species. The results indicated that E2 at high concentrations (50 ng/L and 100 ng/L groups) significantly up-regulated CYP19b gene expression (P0.01) but E2 at low concentration (2 ng/L and 10 ng/L groups) did not. MT had no significant effect on CYP19b expression in early stages, but it significantly inhibited CYP19b expression with prolonged treatment except the relatively high concentrations of MT (50 ng/L group) with the characerisitc of dose correlativity. As androgen analogue, AED was found to have similar effects with MT, and the relatively high concentrations (50 ng/L and 100 ng/L groups) of AED inhibited the CYP19b expression (P0.01). The results indicated the pivotal role of three typical environmental hormones under different concentration and various time points on the expression of CYP19b.