Male rats, neonatally treated with ATD (1,4,6-androstatriene-3,17-dione), which blocks the aromatization of testosterone into estradiol (E2), were tested for adult partner preference behavior (PPB; estrous female vs. active male). Castration caused a decrease in preference for the female partner in all males, with ATD males showing lower preference for the female partner than controls. Long-term castrated males did not show preference for either partner. Precastration levels of PPB in control males occurred after treatment with E2 or dihydrotestosterone (DHT) plus E2. DHT alone had no effect on PPB. With E2 alone, the ATD males clearly preferred the male partner. When DHT was added, these ATD males showed no preference for either partner or a low preference for the female partner. In conclusion, adult PPB in male rats is activated by endogenous testosterone or by both its metabolites (DHT and E2) or by E2 alone. ATD males showed a much lower preference for the female. There was a differential effect of DHT and E2: DHT had no effect, but E2 clearly caused ATD males to prefer the male partner and control males to prefer the female partner.