Objective: Serum concentrations of estradiol (E-2) and testosterone (testo) measured by mass spectrometry-based assays should remain below the 95th centile measured at 9.3 pg/mL for E-2 and 0.26 ng/mL for testo in normal -postmenopausal women in order to avoid the risk of non-physiological systemic exposure to elevated serum concentrations of these two sex steroids. Methods: Serum E-2 and testo, as well as dehydroepiandrosterone (DHEA) and nine of its other metabolites, were measured at 10 time intervals over 24 h on the first and seventh days of daily intravaginal administration of 0.50% (6.5 mg) DHEA by validated mass spectrometry-based assays. Results: No biologically significant change in the individual serum concentrations of E-2, testo or DHEA was observed. Most importantly, estrone sulfate (E-1-S) and the glucuronidated androgen metabolites also remained within normal values, thus confirming the absence of biologically significant systemic exposure in line with intracrinology. Using data from the literature, comparison is made with serum E-2 above normal postmenopausal values following administration of 10-mu g E-2 tablets. Conclusion: While the clinical program on vulvovaginal atrophy has shown the efficacy and safety of intravaginal 6.5 mg of DHEA (prasterone), the present data illustrate in detail the serum levels of the individual sex steroids and their metabolites derived from DHEA. The data obtained are in line with the physiology of intracrinology and confirm an action limited to the vagina as the serum concentrations of all sex steroids are maintained within the normal values of menopause, thus protecting the uterus and most likely other tissues.