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To better understand the mechanisms underlying the beneficial effects of the intravaginal administration of dehydroepiandrosterone (DHEA) observed in postmenopausal women on sexual dysfunction.To identify the distribution of the androgen-synthesizing enzymes as well as androgen receptor (AR) and measure steroid levels in the monkey vagina.The cynomolgus monkey (Macaca fascicularis), the closest model to the human, has been used to measure the expression levels of steroidogenic enzymes and androgen receptor by quantitative reverse transcription polymerase chain reaction (n = 4), confirmed by immunohistochemistry, and immunofluorescence (n = 3). DHEA and its androgenic metabolites were quantified by LC-MS/MS (n = 4).The presence of SRD5A1, SRD5A2, HSD17B3, AR as well as nerve fibers (PGP 9.5) was investigated, and steroid levels were measured.AR is widely distributed within the vaginal epithelium and also in the lamina propria with a lower expression in the muscularis layer and blood vessel walls. Androgen-forming enzymes, on the other hand, are expressed in the vaginal stratified squamous epithelium at a relatively high level where they are uniformly distributed from the basal membrane up to the superficial keratinized cells. The enzymes are at a lower level in blood vessel walls and zona muscularis where nerve fibers are localized. DHEA and its androgen metabolites are present at biologically significant concentrations in the monkey vagina.The enzymes responsible for androgen formation as well as AR are at the highest level in the superficial layer of the stratified epithelium and muscularis layers of the vagina. These data provide a potential explanation for the described role of androgens in regulating vaginal lubrication, smooth muscle activity, blood flow, and the neuronal activity potentially involved in the correction of sexual dysfunction. Bertin J, Dury AY, Ouellet J, Pelletier G, and Labrie F. Localization of the androgen-synthesizing enzymes, androgen receptor, and sex steroids in the vagina: Possible implications for the treatment of postmenopausal sexual dysfunction. J Sex Med 2014;11:1949–1961.