A radioimmunoassay method was utilized to determine serum cortisol (hydrocortisone) concentrations in normal dogs. The mean value was 2.32 .mu.g/dl (minimum, 0.94; maximum, 3.70; with a coefficient of variation of 29.7%). Samples were collected in the morning of the diurnal cycle. The cortisol antibody used was highly specific for cortisol and showed little cross reaction with other common physiologic corticosteroids, when they were assayed using the radioimmunoassay procedure described.