The cholesterol catabolic pathway occurs in most mycolic acid-contg. actinobacteria, such as Rhodococcus jostii RHA1, and is crit. for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl-CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild-type and accumulated 3aα-H-4α(3’-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), consistent with the catabolism of half the steroid mol. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3Mtb catalyzed the ATP-dependent CoA thioesterification of HIP and its hydroxylated analogs, 5α-OH HIP and 1β-OH HIP. The apparent specificity const. (kcat/Km) of FadD3Mtb for HIP was 7.3 ± 0.3 × 105 M-1s-1, 165 times higher than for 5α-OH HIP, while the apparent Km for CoASH was 110 ± 10 μM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3Mtb did not detectably transform a metabolite with a partially degraded C17 side-chain. Overall, these results indicate that FadD3 is a HIP-CoA synthetase that initiates catabolism of steroid rings C and D after side-chain degrdn. is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degrdn. enzymes. [on SciFinder(R)]