A short chain enoyl-CoA hydratase (crotonase) from pig heart has been purified to apparent homogeneity. The enzyme has an estimated native molecular weight of 155,000 and appears to be composed of six subunits of molecular weight 27,300. A study of the kinetic properties of the enzyme revealed that the maximal velocity decreases nearly linearly with increasing chain length of the substrates from 1,670 units/mg with crotonyl-CoA to 40 units/mg with hexadecenoyl-CoA. However, the same Km values of 30 muM were obtained for all substrates except for crotonyl-CoA for which a value of 13 muM was determined. Since the presence of both crotonase and long chain enoyl-CoA hydratase in pig heart has been reported earlier, the presence of the same two enoyl-CoA hydratases in various tissues of several animals was investigated by sequential extraction and chromatography on hydroxylapatite of tissue homogenates. The simultaneous occurrence of both types of enoyl-CoA hydratase in tissues of pig and guinea pig has thus been established. It is proposed that the complementary actions of the two enoyl-CoA hydratases assures a high rate of hydration of enoyl-CoA intermediates of all chain lengths in fatty acid oxidation.