Sex preference in male rats is partly determined by the organizational action of estradiol. Thus, several paradigms have used aromatase inhibitors to manipulate sex preference. We recently showed that a subpopulation of male rats prenatally treated with letrozole (0.56 μg/kg, G10–G22), a non-steroidal third generation aromatase inhibitor, had same-sex preference, female sexual behavior (including lordosis and proceptivity) and penile erections when exposed to other males. These males, in addition, displayed high levels of experimental anxiety in the plus maze test and were insensitive to the anxiogenic-like acute effect of FLX (10 mg/kg). The two main purposes of the present work were: a) to study the behavioral profile of males displaying same-sex preference in the forced swim test (FST), and b) to analyze if the antidepressant-like effect of the subchronic treatment with FLX (10 mg/kg, 3 times) or desipramine (DMI, 10 mg/kg, 3 times) vary according to sex preference. Males treated prenatally with letrozole with same-sex preference showed more immobility and less active behaviors (swimming and climbing) in the FST than males with female preference. Subchronic treatment with FLX and DMI reduced immobility when compared to saline controls, while FLX increased swimming and DMI increased climbing behavior. Treatments were equally effective in males with preference for other males and those that preferred females. These results indicate that an association exists between prenatal letrozole treatment, same-sex preference and immobility in the FST.