The objective of this study was to contribute to investigate the efficacy of letrozole, a potent non-steroidal aromatase inhibitor, on serum sex steroid levels and oocyte growth in rainbow trout females. Serum steroid levels were measured using an indirect enzyme-linked immunosorbent assay (ELISA) before and 22 days after an initial injection with 1, 2.5 mg kg-1 and subsequent weekly injection with 2.5 mg kg-1letrozole. After the last blood collection, gonad tissues were removed and oocyte diameter and GSI were determined. In all groups treated with letrozole, serum 17β- esteradiol levels (E2) decreased significantly and inversely serum 17α-20ß dihydroxy-4 pregnen- 3- one (17α-20ß-P), testosterone(T) levels increased significantly from 6 to 96 h after injection (p < 0.05). The lowest E2 levels, oocyte diameter and GSI were measured in the group treated weekly with 2.5 mg kg-1 letrozole (p < 0.05). This study demonstrates that letrozole has the potential to interfere in the synthesis of endogenous estrogens from androgens and delay gonadal development.