ABSTRACT: Comamonas testosteroni TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by β-oxidation. In this study, we revealed that 7β,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3S)-3-hydroxylacyl CoA-dehydrogenase, encoded by scdE (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by scdF (ORF23). With these results, the whole cycle of β-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1