Male rats were neonatally treated with 1,4,6-androstatriene-3,17-dione (ATD), which blocks aromatization of testosterone (T) to estradiol (E2), from Days 0, 2, or 5 through 14. Adult partner preference behavior (PPB; choice between estrous female rat [F] and active male rat [M]) was studied in the early part of the dark phase of the light-dark (LD) cycle. ATD Day 0 Ms showed a preference for the stimulus M or showed no preference for either of the stimulus Ss. Controls preferred the estrous F. ATD Days 2 and 5 Ms showed PPB intermediate between ATD Day 0 Ms and controls. Thus the neonatally sensitive period for organization of adult PPB extends beyond Day 5. Furthermore, PPB showed a nocturnal rhythmicity in ATD Ms but not in controls. In the late part of the dark phase, all Ms showed a preference for the stimulus F. ATD Days 2 and 5 Ms and control Ms were no longer different in PPB, but ATD Day 0 Ms still showed significantly lower preference scores for F than all other Ms. Thus the E2 metabolite of T suppresses organization of an adult nocturnal rhythm in PPB.