A chromatographic procedure that allows measurement of [3H]prednisone and [3H]prednisolone in biologic fluids and tissues was developed and used to determine some of the biochemical properties of prednisone and its distribution and metabolism in mice, dogs, and monkeys. When prednisone was added to mouse serum in vitro at a concentration of 50 microgram/ml, approximately half of the drug became bound to serum proteins. Homogenates of mouse liver metabolized prednisone to prednisolone. Following ip or oral doses of prednisone to mice, serum levels of prednisone, prednisolone, and other metabolites were maximum at 15 minutes. 20-Dihydroprednisolone was identified as a metabolite in several biologic samples. For dogs and monkeys given in iv dose of prednisone, disappearance from the serum was biphasic with apparent half-lives of 15 and 82 minutes and 33 and 78 minutes respectively. The serum levels of prednisone and prednisolone in mice, dogs, and monkeys were not increased beyond expected levels by simultaneous administration of iv doses of vincristine. The prednisone content of the kidneys, liver, intestine, and bile of a monkey killed 30 minutes after an iv dose was higher than that in serum. Oral doses of prednisone were absorbed erratically by dogs and monkeys. Only small amounts of prednisone and prednisolone were excreted in the urine of dogs and monkeys given an iv or oral dose.