The metabolic fate of 20α-hydroxypregn- 4-en-3-one (20α-OH-P) in surviving rabbit liver tissue slices was determined by use of the steroid labeled with tritium in the 20β-position and with C14 at position 4. After incubation periods of 30 and 120 min in Krebs-Ringer buffer, the C14-labeled metabolites were fractionated and the retention of 3H by each was determined. Tritium which had been removed from 20α-OHP did not contribute significant radioactivity to subsequently reduced steroid metabolites. After 30 min of incubation, the 20α-OH-P had been 84% metabolized to other products, but the remaining precursor had lost 13% of its tritium. Pregnane-3a,20α-diol, a major metabolite of 20α-OH-P and of progesterone in the rabbit liver, retained 95% of the tritium of the precursor; thus, very little 20α-OH-P was converted to progesterone during the course of its metabolism. The pregnane-3,6,20-triols had lost 50% of the 3H originally at C-20β. C-20 oxidation occurred primarily after 6-hydroxylation, indicating the presence of a 20α-hydroxysteroid oxidoreductase specific for 6-hydroxysteroids. Formation of organic acid esters of progestin metabolites by liver tissue has been described for the first time; these are peculiar to metabolism of 20α-OH-P, and have not been previously found with progesterone as the substrate. The evidence favors the concept of separate metabolic routes for the metabolism of 20α-OH-P and progesterone in rabbit liver. (Endocrinology88: 249, 1971)