The role of oestrogens and androgens on bone metabolism has been studied extensively. However, less is known about the effects of progestins. The progesterone receptor (PR) is expressed in human osteoblasts. Progestins have been found to stimulate osteoblast proliferation, differentiation and growth factor expression (e.g. IGFBP-5) and to increase bone formation. In postmenopausal women progestins combined with oestrogen increased bone density to a greater extent than oestrogen alone. We have postulated that pre-receptor regulation by metabolising enzymes may have a major impact upon PR-mediated changes in bone turnover. We have therefore investigated the metabolism of progesterone in primary cultures of human osteoblasts from 7 patients. Sub-confluent monolayers were incubated in triplicates for 16h with 4-14C-progesterone (200,000cpm) and 50nM unlabeled progesterone. Steroids were extracted with dichloromethane and run with two-dimensional thin-layer chromatography (TLC) for identification. The TLC plates were scanned using a BioRad Molecular Imager Fx. In parallel RNA was extracted and RT-PCR analysis carried out to identify progesterone metabolizing enzymes. Intensive progesterone metabolism was observed in all cultures (up to 40% in 16h) with the activity being of the order of 1pmol/mg protein/h. The two major products were the inactive 20alpha-DH-progesterone and 5alpha-DH-progesterone. We detected high levels of mRNA for the enzyme AKR1C1 and 5alpha-reductase type1, which are responsible for those conversions. In addition we found the ring A reduced metabolites 3alpha,5alpha- and 3beta,5alpha-TH-progesterone and their 20-reduced products. The 3alpha-reduction is performed by AKR1C2, and the 3beta-reduction either by 3beta-HSD type 1 or by hydroxysteroid-epimerase. In conclusion we have demonstrated a striking capacity for inactivation of progesterone in human osteoblasts which could, in turn, regulate the access of progesterone to its receptor. Alterations in enzyme expression, especially of AKR1C1 and 5alpha-reductase type 1, could therefore have a major impact on progesterone-mediated effects on osteoblast proliferation, differentiation and bone formation.