ABSTRACT The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc ₁ complex and terminating in the aa ₃ -type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc ₁ complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB , which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd -type menaquinol oxidase that is required for the growth of M. smegmatis under O ₂ -limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd -type oxidase were revealed by expression profiling of the bc ₁ -deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc ₁ - aa ₃ pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.