A radioimmunoassay for plasma cortisol (hydrocortisone) was developed and validated for sensitivity, specificity, accuracy, precision, and parallelism. Steroids were extracted with ethyl ether, and cortisol was purified by gel column chromatography prior to assay. [1,2-3H] cortisol and a commercially available sheep antibody to cortisol-21-hemisuccinate were used. Free steriods were separated from bound steroids by centrifugation after adsorption to dextran-coated charcoal. Plasma cortisol was measured by this technique in 6 normal dogs. Circadian rhythm of cortisol secretion was not detected in samples obtained by venipuncture at 8 different hours on 3 separate days, suggesting that adrenal function tests may be started in clinical patients at any time of day. Resting plasma cortisol concentrations averaged 19.4+/-3.0 (SD) ng/ml and ranged from nondetectable (less than 3 ng/ml) to 77.5 ng/ml. Of 144 canine plasma samples, 95% contained less than 50 ng of cortisol/ml. Intramuscular injection of 2.2 units of adrenocorticotropic hormone/kg of body weight caused detectable increase in plasma cortisol concentrations; maximum response (68.3 to 111.6 ng/ml) occurred 1 to 2 hours after injection. Oral administration of dexamethasone suppressed plasma cortisol to nondetectable concentrations for 32 hours in all 6 dogs.