A radioimmunoassay for measurement of cortisone in plasma has been developed utilizing rabbit anti-sera against a cortisone-bovine serum albumin complex. To insure specificity thin layer chromatography was used because unchromatographed samples resulted in 10–20% higher values than the chromatographed, even though the cross reactivity with cortisol was only 0.68% and with corticosterone 1.15%. The sensitivity of the assay varies 50–79 pg with recovery of cortisone added to plasma found to be 94.2 ± 5.8 (sd). The mean morning plasma level of cortisone in young men was 2.6 μg/100 ml ± 0.5 (sd) and in women 1.96 ± 0.54. Evening levels decreased in relation to cortisol concentration. Pregnant women had elevation of cortisone levels comparable to that of cortisol. After dexamethasone suppression plasma cortisone levels decreased but following ACTH and usually during acute stress there were no significant changes noted. In patients with severe liver disease cortisone concentration tended to be lower than normal but directly proportional to cortisol levels whereas in patients with kidney disease cortisone concentration was subnormal. Two patients with Cushing's syndrome had elevated levels of cortisone comparable to cortisol but after o,p′DDD therapy the concentration of cortisone decreased disproportionately thus suggesting an inhibitory effect by the drug on conversion of cortisol to cortisone. The results indicate that circulating cortisone is formed mainly in peripheral tissues with the kidney being a major source.