A sensitive and reliable radioimmunoassay for urine and plasma 6beta-hydroxycortisol has been developed. Antiserum showing high specificity against 6beta-hydroxycortisol was produced in rabbits immunized with 6beta-hydroxycortisol 21-hemisuccinate-bovine serum albumin. The sensitivity of the assay was 25 pg on a diluted sample equivalent to 1 microliter of urine, and on 50 microliter of plasma after separation by celite chromatography. The intra- and inter-assay coefficients of variation for urine were 4.8 and 6.7% and those for plasma were 4.2 and 12.1%. Concentrations were determined in patients with bronchogenic carcinoma, in patients treated with dilantin, in neonates, and in infants aged 5-12 months.