Masculinization is programmed by androgen exposure during a masculinization programming window (MPW). Deficiency in MPW androgen action results in reduced size of all reproductive organs and anogenital distance (AGD) and reproductive disorders. Although timing of MPW closing has been defined, what determines 'opening' and 'closing' of the MPW remains unknown. To test whether initiation of testosterone production/action defines the opening of the window, we first demonstrated that androgen receptor mRNA and protein are expressed prior to the MPW, and then investigated whether masculinization could be advanced or enhanced by treating pregnant rats with either 1 or 10 mg/kg/day dihydrotestosterone (DHT) prior to (early window, EW; e11.5-e14.5) or during the MPW (e15.5-e18.5), and then evaluating offspring in foetal life (e18.5, e21.5), early puberty (day 25) or adulthood (∼day 75). DHT treatment did not affect pregnancy duration, birth, litter or pup size. DHT exposure in either time window did not advance foetal male development (Wolffian duct coiling) and had no effect on AGD, testis, penis and ventral prostate (VP) size at any age when measured; there was a tendency towards smaller penis size. In contrast, exposure of females to 10 mg DHT in either time window induced varying degrees of masculinization, including stabilization of the Wolffian duct and increased AGD (e21.5, Pnd25), VP formation, more male-like phallus structure, absence of nipples and vaginal opening and, in some adult females, gross fluid distension of the uterus (hydrometrocolpos); these effects were generally more pronounced after exposure in the MPW than in the EW. In conclusion, exposure of the male rat foetus to additional androgens prior to or during the MPW does not advance or enhance any measured parameter of reproductive development. Therefore, androgen availability plays no role in determining timing of the MPW. Susceptibility of the female reproductive system to androgens may precede the MPW.