Endometriosis is a widespread condition affecting approximately 10% of women of reproductive age worldwide (Eskenazi & Warner, 1997). Clinical studies have demonstrated the effectiveness of the progesterone receptor (PR) antagonist mifepristone (RU486), in the treatment of this disease (Kettel et al, 1996). The aim of this work was to establish a native tissue binding assay for determining the potency and ex-vivo receptor occupancy (RO) of PR antagonists in the rabbit. With Pfizer ethical approval, sexually immature New Zealand White rabbits (700-900g) were dosed daily (q.d) for 2weeks with 0.1mg/kg ethinyl estradiol. Animals were killed and snap frozen uterine tissues used for either homogenate binding (Issar et al., 2006) or autoradiography (ARG), adapted from Brown et al.,1995. Cryostat sections (20μM) or cytosolic tissue fractions (160µl) were incubated with 1.5nM [3H]R5020 (Perkin Elmer, UK) in the absence (total binding) or presence (non-specific binding) of RU486 (10µM). Dried tissue sections were imaged using a BetaImager for 8h. In homogenate binding experiments, bound and unbound fractions were separated via charcoal centrifugation. For IC50 determination, sections were incubated with a range of concentrations (10pM–10µM). Specific binding (counts/mm2) was quantified using Betavision and IC50 analysis performed using an in-house curve fitting programme. Following optimisation experiments to minimise incubation times, ex-vivo RO studies were performed comparing untreated animals with animals dosed q.d. with RU486 (3mg/kg) for 5 consecutive days. Animals were sacrificed 1h post-dose. Data are expressed as mean ± standard error of the mean. Both homogenate binding and ARG produced specific binding >70% at PR. RU486, progesterone and dexamethasone inhibited [3H]R5020 binding with IC50 values of 16.6±2.3nM, 57.2±11.7nM and >1μM respectively (n=4). In contrast to the reported 18h incubation for in-vitro studies (Brown et al.,1995), using our optimised conditions, we found no difference in % specific binding after 18h or 30min and therefore selected this incubation time for all subsequent ex-vivo RO studies. In tissue sections from rabbits dosed with RU486 (3mg/kg), an effective dose in the rabbit McPhails test of endometrial arborisation (Elger et al, 2000), a mean RO of 74±3% (n=4) was calculated. In summary, this study has shown that ARG is a suitable method for determining the binding affinity of PR ligands in native rabbit endometrium. In addition, we have shown for the first time, optimised binding conditions for an ex-vivo RO assay for PR. The RO data obtained with RU486 in this pre-clinical model may be used as a benchmark to profile other PR ligands to build confidence in the prediction of clinically efficacious doses.