Five subcellular fractions were obtained by successive centrifugation from the liver of rats within 6 hours of life and characterized by comparing marker compound or marker enzyms. After incubating 3β-hydroxy-5α-pregnan-20-one with the each fraction, the steroids were analyzed by TLC, GLC and GC-MS. A 6α-hydroxylase which hydroxylizes the tetra-hydrogenated compound of progesterone, 3β-hydroxy-5α-pregnan-20-one, was localized in the crude plasma membrane fraction, but not in the microsome fraction. The maximum 6α-hydroxylation was observed at pH 7.0. While this 6α-steroid hydroxylase was not able to hydroxlyze the progesterone, the 3α-isomer was hydroxylized at the 6α-position.