The Rhodococcus erythropolis SQ1 kstD3 gene was cloned, heterologously expressed and biochemically characterized as a KSTD3 (3-keto-5α-steroid Δ¹-dehydrogenase). Upstream of kstD3, an ORF (open reading frame) with similarity to Δ⁴ KSTD (3-keto-5α-steroid Δ⁴-dehydrogenase) was found, tentatively designated kst4D. Biochemical analysis revealed that the Δ¹ KSTD3 has a clear preference for 3-ketosteroids with a saturated A-ring, displaying highest activity on 5α-AD (5α-androstane-3,17-dione) and 5α-T (5α-testosterone; also known as 17β-hydroxy-5α-androstane-3-one). The KSTD1 and KSTD2 enzymes, on the other hand, clearly prefer (9α-hydroxy-)4-androstene-3,17-dione as substrates. Phylogenetic analysis of known and putative KSTD amino acid sequences showed that the R. erythropolis KSTD proteins cluster into four distinct groups. Interestingly, Δ¹ KSTD3 from R. erythropolis SQ1 clustered with Rv3537, the only Δ¹ KSTD present in Mycobacterium tuberculosis H37Rv, a protein involved in cholesterol catabolism and pathogenicity. The substrate range of heterologously expressed Rv3537 enzyme was nearly identical with that of Δ¹ KSTD3, indicating that these are orthologous enzymes. The results imply that 5α-AD and 5α-T are newly identified intermediates in the cholesterol catabolic pathway, and important steroids with respect to pathogenicity.