Pejerrey is a teleost fish presenting a strong temperature-dependent sex determination. This study was conducted to clone pejerrey amh cDNA, analyze its expression profile during thermal and endocrine manipulation of gonadal differentiation, and compare its expression with that of gonadal aromatase (cyp19a1). Amh displayed higher expression at masculinizing than at feminizing temperatures during the gonadal differentiation period. Its expression at an intermediate temperature (females 1:1 males), was high in half of the larvae and low in the other half. Cyp19a1 showed a reciprocal expression pattern to that of amh both individually- and temperature-wise. Increased cyp19a1 and amh expression was observed before morphological gonadal differentiation. Amh expression in larvae feminized by administration of estradiol or masculinized by the administration of an aromatase inhibitor was down- and up-regulated, respectively. These results show that amh plays a critical role in testicular differentiation and it is apparently modulated by estrogens in this species. Developmental Dynamics 237:3192–3199, 2008. © 2008 Wiley-Liss, Inc.