Di-(2-ethylhexyl) phthalate (DEHP) interferes with male reproductive endocrine system in mammals, however its effects on fish reproduction are largely unknown. We evaluated sperm quality and investigated reproductive endocrine system in mature goldfish (Carassius auratus) exposed to nominal 1, 10, and 100 mu g/L DEHP. To examine DEHP estrogenic activity, one group of goldfish was exposed to 17 beta-estradiol (5 mu g/L E-2) for comparison. Following 30 d of exposure, sperm production was decreased and suppressed in DEHP and E-2 treated goldfish, respectively. Sperm motility and velocity were decreased in goldfish exposed to 100 and 10 mu g/L DEHP at 15s post-sperm activation, respectively. Compared to control, 11-ketotestosterone (11-KT) levels were decreased at 10 and 1 mu g/L DEHP at day 15 and 30, respectively. In E-2 treated goldfish, 11-KT levels were decreased compared to control during the period of exposure. E-2 levels were increased in goldfish exposed to E-2, but remained unchanged in DEHP treated goldfish during the period of exposure. StAR mRNA levels encoding regulator of cholesterol transfer to steroido-genesis were decreased in DEHP and E-2 treated goldfish following 15 and 30 d of exposure, respectively. Luteinizing hormone (LH) levels were decreased in DEHP and E-2 treated goldfish following 15 and 30 d of exposure, respectively. In DEHP treated goldfish, gnrh3, kiss) and its receptor (gpr54) mRNA levels did not change during the experimental period. In E-2 treated goldfish, gnrh3 mRNA levels were decreased at day 7, but kiss1 and gpr54 mRNA levels were increased at day 30 of exposure. The mRNA levels of genes encoding testicular LH and androgen receptors remained unchanged in DEHP and E2 treated goldfish. In contrast to E2 treated goldfish, vitellogenin production was not induced in DEHP treated goldfish and mRNA levels of genes with products mediating estrogenic effects remained unchanged or decreased. In conclusion, DEHP interferes with testis and pituitary hormonal functions to reduce sperm quality in goldfish and does not exhibit estrogenic activity.