It is now generally acknowledged that local tissue concentrations of cortisol and cortisone are modulated by site-specific actions of 11β-hydroxysteroid dehydrogenase (11β-HSD) isoenzymes 1 and 2. Cortisone, the inactive metabolite of cortisol is produced by type 2 11β-HSD. To assess 11β-HSD type 1 and 2 activity, the cortisone/cortisol ratio has to be accurately determined. Immunoassays to measure cortisone levels are not widely available and tend to lack specificity. The aim of this project was to develop a highly specific and sensitive ELISA method for the estimation of free cortisone levels in urine, saliva and in vitro media samples without chromatographic separation. Antibodies against cortisone were raised in rabbits by using cortisone-3-CMO-KLH as immunogen. HRP-goat anti-rabbit IgG conjugate (Upstate, UK) was used as enzyme tracer. Urine and cell incubation media samples were either extracted with dichloromethane or assayed directly following a 10× dilution. Saliva were extracted with ether and reconstituted in assay buffer. Aliquots were then assayed using the ELISA technique previously described after minor modifications (Al-Dujaili et al. 2009 Steroids 74 456462). Cross-reactivities of the untreated cortisone antisera with major potential interfering steroids were minimal except for cortisol (3.15%). However, following an immunoaffinity purification of the antibodies (incubation with CNBr-activated sepharose-cortisol-3-CMO-BSA for 24 h), cross reactivity of the purified cortisone antibody with cortisol was reduced to 0.6%. Minimum detection limit of cortisone ELISA was 28 pg/ml (77.7 pmol). The validity of the cortisone ELISA was confirmed by the good correlation obtained before and after an HPLC fractionation step. Inter-assay imprecision was 8.712.8% CV. Using this assay, salivary cortisone levels showed a circadian rhythm (11.2±7.3 nmol at 0800 h and 5.1±3.6 nmol at 1800 h, n=20), and the levels were reduced following liquorice ingestion. In adrenal H295 cell line incubations, basal cortisone levels were 4.24±0.22 nmol that went up to 7.6±2.2 nmol post forskolin stimulation. Urinary free cortisone excretion in healthy volunteers was 56.66±36.9 nmol/day, n=32). In human volunteers following ingestion of green coffee bean extract for 2 weeks, urinary free cortisone excretion reduced significantly from 58.38±9.3 to 34.74±4.9 nmol/day (P=0.016) and cortisone/cortisol ratio (1.44±0.33to 0.96±0.21). In conclusion, a simple and highly specific and sensitive ELISA has been developed to estimate cortisone levels in biological fluids.