It generally is held that the murine vagina develops from the urogenital sinus and the lower portion of the Müllerian ducts and that both endodermally derived sinus epithelium and mesodermally derived Müllerian epithelium contribute to the adult vagina. We tested Müllerian and urogenital sinus-derived vaginal epithelia for their ability to differentiate in response to various hormonal conditions and compared these responses to those of the in situ vagina. Tissue recombinants were prepared with 0-day Müllerian-derived vaginal epithelium and vaginal mesenchyme. Similarly, tissue recombinants containing urogenital sinus-derived vaginal epithelium were prepared with either 0-day sinus vaginal epithelium or 16-day fetal urogenital sinus epithelium and vaginal mesenchyme. Müllerian- or sinus-derived vaginal mesenchyme was used to construct the tissue recombinants; however, the source of the mesenchyme had no influence on the results. Tissue recombinants were grafted to the renal capsule of female mice, allowed to develop for 1 month, and exposed to various hormonal treatments. In diethylstilbestrol-treated hosts, all tissue recombinants regardless of the source of the epithelium were lined by a cornified epithelium. In contrast, only in tissue recombinants containing mesodermally derived Müllerian vaginal epithelium did the epithelium mucify in response to progesterone plus estrogen or become atrophic in ovariectomized hosts. These are the same epithelial modifications seen in the hosts' vagina. Tissue recombinants containing endodermally derived urogenital sinus epithelium or sinus vaginal epithelium and grafts of intact urogenital sinus maintained a stratified squamous noncornified epithelium in both ovariectomized and progesterone plus estrogen-treated hosts. Furthermore, in tissue recombinants containing Müllerian vaginal epithelium and vaginal mesenchyme, estrogen induced a slightly higher epithelial labeling index than in tissue recombinants containing urogenital sinus epithelium or sinus vaginal epithelium. The epithelial labeling index was the same regardless of the source of the vaginal mesenchyme. These results indicate that Müllerian-derived and sinus-derived vaginal epithelia are not equivalent and suggest that Müllerian vaginal epithelium displaces sinus vaginal epithelium during postnatal development. The replacement may result in part from a slight but consistently higher proliferation rate in Müllerian versus sinus vaginal epithelium.