Masculine brain sexual differentiation in rodents is due to testosterone conversion to estradiol by the aromatase complex, producing males that when adults display their typical sexual behavior, like partner preference towards the receptive female. Disruption in endocrine milieu during development by the administration of the aromatase inhibitor, letrozole (56 μg/kg/day gestational days 10-22) results in 30% of males with same-sex preference, while the rest retain preference for the receptive female. The males with same-sex preference showed high levels of experimental anxiety in the elevated plus maze test. In addition, independently of the partner preference, this letrozole treatment produced an earlier preputial separation, and feminization of the second and fourth digit length index and of the length of some pelvic bones. These results suggest that alterations in the hormonal levels of estradiol during development not only affect the central nervous system, but also other biological markers sensitive to androgen concentrations.