The genes controlling the biosynthesis of the carotenes in Mycobacterium aurum were clustered in a 10.83-kb segment. Fragments generated by endonuclease digestions of the segment were cloned into a pHLD69 shuttle vector. The plasmids so constructed were used to transform a colorless (albino) M. aurum mutant (strain A11), a brick-red mutant accumulating large amounts of lycopene (strain NgR9), the buff-colored Mycobacterium smegmatis MC2-155, and the buff-colored Mycobacterium tuberculosis H37Ra. From the endonuclease digestion patterns and the phenotypes of the transformed strains, the partial physical and functional maps of a carotenogenesis operon were established. This investigation also showed that the genes controlling the conversion of lycopene into the xanthophylls were not located in the 10.83-kb segment.