Abstract An investigation by high-performance liquid chromatography (HPLC) of the metabolites from the livers of cows and steers treated with [4- 14C]-progesterone revealed them to consist principally of 3α-hydroxy-5β-pregnan-20-one and 5β-pregnane-3α,20β-diol. In bovine kidney tissue the principal metabolites were found to be 20β-hydroxypregn-4-en-3-one, 3α and 3β-hydroxy-5α-pregnan-20-one and 5α-pregnane-3β,20β-diol in about equal amount, plus about 15% of the total as unconverted progesterone. In milk the major product was 5α-pregnane-3,20-dione in addition to about 80% of the total as unconverted progesterone. Only traces of compounds other than progesterone were found in kidney fat. These metabolites were further characterized by crystallization to constant specific acitivity, including derivative formation. This study demonstrates for the first time the utility of HPLC for the rapid analysis of a complete mixture of metabolites of progesterone from crude extracts of mammalian tissues.