Dehydroepiandrosterone (DHEA) sulfate (DHEAS) is the most abundant steroid in the human circulation and is thought to be the circulating hydrophilic storage form of DHEA. It is generally accepted that DHEA and DHEAS inter-convert freely and continuously via hydroxysteroid sulfotransferases and steroid sulfatase and that only desulfated DHEA can be converted downstream to sex steroids. Here we analyzed DHEA/DHEAS interconversion in vivo and in vitro. We administered oral DHEA (100 mg) and iv DHEAS (25 mg) to eight healthy young men, resulting in similar increases in serum DHEAS compared with baseline. However, although DHEA administration significantly increased serum DHEA (P < 0.05), no such increase was observed after DHEAS. Similarly, DHEA but not DHEAS was converted downstream to androstenedione, estrone, and androstanediol glucuronide. The striking absence of conversion of DHEAS to DHEA was mirrored by our in vitro findings in HepG2 cells, revealing dose-dependent conversion of DHEA (0.1-2 mum) to DHEAS but no conversion of DHEAS (0.1-2 mum). These results clearly illustrate a lack of hepatic conversion of DHEAS to DHEA, challenging the concept of free interconversion of DHEA and DHEAS. DHEAS does not seem to represent a circulating storage pool for DHEA regeneration, and therefore serum DHEAS is unlikely to reflect bioavailable DHEA.