Clinical isolates of the opportunistic pathogen Mycobacterium avium complex (MAC) undergo a reversible switch between red and white colony morphotypes on agar plates containing the lipoprotein stain Congo red. Compared to their isogenic red counterparts, white morphotypic variants are more virulent and more resistant to multiple antibiotics. This report shows that the two-component regulatory system mtrAB is required for the red-to-white switch as well as for other morphotypic switches of MAC. A mutant with a transposon insertion in the histidine protein kinase gene mtrB was isolated from a morphotypically white parent clone. The mutant resembled a naturally occurring red morphotypic variant in that it stained with Congo red, was sensitive to multiple antibiotics, and was permeable by a fluorescent DNA stain. However, it differed from a red variant in that it could not switch to the white or transparent morphotype, and it could not survive intracellularly within macrophage-like cells. Transcomplementation with a cloned wild-type mtrB gene restored to the mutant the ability to form impermeable, drug-resistant white and transparent variants. Quantitative reverse transcriptase PCR showed that mtrB was required for the normal expression of cell surface Mce proteins, some of which are up-regulated in the red-to-white switch. The results indicate that mtrAB functions in regulating the composition and permeability of mycobacterial cell walls and plays a role in the reversible colony type switches of MAC.