Inhaled corticosteroids are the drugs of choice for asthma and other airway allergy diseases (Barnes et al, 2004). Despite direct delivery into the lungs inhaled corticosteroids are absorbed into the systemic circulation with the potential to cause side effects (Barnes et al, 2004). In this study we evaluated [3H]Dexamethasone (DM) as a suitable radioligand for assessing glucocorticoid receptor (GR) occupancy in lung and side effect tissues. With local ethical approval, Sprague-Dawley rats (250-350g) were sacrificed by decapitation. Lung, thymus, liver, kidney and brain were resected and frozen in isopentane. Saturation experiments were performed by incubating cytosolic fraction of tissue homogenate with 0.1-40nM [3H]DM (Perkin Elmer,UK) in the absence (total binding) or presence (non-specific binding) of DM (1µM) for 3h at 4oC. Bound and unbound fractions were separated via charcoal centrifugation (Hochhaus, 1995) or rapid filtration through GF/B filters. Following ex vivo optimisation studies, fluticasone propionate (FP) receptor occupancy (RO) was measured by comparing control animals with animals dosed by the intratracheal route with 100µg of FP nanosuspension at 1,2,4 and 8h post dose. Data are expressed as mean±standard error of the mean. In vitro, [3H]DM bound with high affinity with specific binding >80%. Saturation analysis showed [3H]DM had similar affinity for the GR expressed in tissues examined (5.4–12.4nM), consistent with previous studies using [3H]DM binding in rat liver (Kenji et al, 1985.) and human lung (Philip et al, 1974). However, levels of GR expression varied between tissues with Bmax (mean fmol/mg protein±s.e.mean) rank order of thymus(267±10)>liver(228±31)>lung(136±26)>kidney(91±10)>brain(44±4). Time dependant decrease in ex vivo RO was seen in lung and liver following 100µg intratracheal dosing of FP with no difference in RO values comparing either 3h or 18h incubation or filtration versus charcoal separation. Based on this data we selected 3h incubation time and filtration for all subsequent ex vivo RO studies. In contrast, previous ex vivo assays have been performed using [3H]triamcinolone with 18h incubation time using charcoal centrifugation (Hochhaus et al, 1995). Further ex vivo time course studies with FP demonstrated consistently higher RO in lung compared to liver. Interestingly, similar RO values to the lung were also observed in the thymus. In summary we have reported an improved way of measuring ex vivo GR occupancy by reducing incubation time with [3H]DM and using filtration methods. This may allow us to relate pharmacological responses to RO in various tissues in pre clinical models of asthma and differentiate compounds based on their therapeutic index.