Anterior pituitary glands from diestrous and proestrous rats were incubated with 3H-progesterone. After subcellular fractionation, the nuclear pellet contained 25-29% of the radioactivity whereas 30-33% of the radioactivity was associated with the 1000 × g supernatant. By reverse isotopic dilution, the radioactivity extracted from both diestrous and proestrous nuclear fractions was characterized as progesterone, 5α-pregnane- 3,20-dione and 3α-hydroxy-5α-pregnan-20- one. Approximately 60-65% of the radioactivity remained as progesterone but 5-10% was identified as 5α-pregnane-3,20-dione, and 10-20% as 3α-hydroxy-5α-pregnan-20-one. Of the radioactivity extracted from both diestrous and proestrous 1000 × g supernatants, 35% remained as unaltered progesterone and 15% was identified as 3α-hydroxy- 5α-pregnan-20-one. Less than 1% of the radioactivity in the 1000 × g supernatant remained congruent with 5α-pregnane-3,20-dione. (Endocrinology93: 430, 1973)