Previous experiments combining the effects of exogenous oestrogens, tamoxifen (antioestrogen) and temperature have implicated oestrogens in the sexual differentiation of gonads in Emys orbicularis, a turtle species with temperature-dependent sex determination (TSD). This has now been extended by examining how two non-steroidal aromatase inhibitors, CGS 16949A and CGS 20267, affect gonadal differentiation in this species. In vitro, ovarian aromatase activity was halved by CGS 16949A at 2.5 · 10−9M and by CGS 20267 at 1 · 10−9M. Several experiments were carried out in vivo: 0.5–20 μg of either inhibitor was deposited onto the eggshell before and two or three times during the thermosensitive period for gonadal differentiation. The effects of treatment were examined at the end of embryonic life. Neither aromatase inhibitor disturbed testis differentiation at a male-producing temperature of 25° C. At a female-producing temperature of 30° C, aromatase inhibition did not give an all-or-none response but different degrees of gonadal masculinization. In some individuals, gonads had the appearance of ovaries but the medulla presented thin epithelial cords and/or large lacunae bordered by a flat epithelium. In other individuals, gonads were ovotestes or testes. CGS 20267 was more potent in masculinizing gonads than CGS 16949A. Indeed, 35% of individuals treated with CGS 20267 had ovotestes or testes whereas only one individual treated with CGS 16949A had ovotestes. The masculinization by aromatase inhibitors of gonads at a female-producing temperature confirms the implication of oestrogens and concords with a key role for aromatase activity in gonadal differentiation of E. orbicularis.