Mycobacterium tuberculosis grows within the phagocytic vacuoles of macrophages, where it encounters a moderately acidic and possibly nutrient-restricted environment. Other mycobacterial species encounter acidic conditions in soil and aquatic environments. We have evaluated the influence of pH and divalent cation levels on the growth of M. tuberculosis and seven other mycobacterial species. In a defined medium, the growth of M. tuberculosis was very restricted by acidic pH. Higher levels of Mg(2+) were required for growth of M. tuberculosis in mildly acidic media (pH 6.0 to 6.5) compared to pH 7. 0 medium. The divalent cations Ca(2+), Zn(2+), or Mn(2+) could not replace Mg(2+) during growth at pH 6.25, but Ca(2+) could at least partially substitute for Mg(2+) during growth at pH 7.0. Among eight species of mycobacteria tested, there was a diversity of growth rates in media with acidic pH and low Mg(2+) levels. M. tuberculosis was the most restricted in growth at pH 6.0, and all of this growth required elevated levels of Mg(2+). M. kansasii and M. smegmatis also grew very poorly in acidic media with limiting Mg(2+). M. fortuitum, M. marinum, M. scrofulaceum, M. avium, and M. chelonae grew at pH 6.0 in an unrestricted manner. These results demonstrate that M. tuberculosis is unique among the mycobacteria in its extreme sensitivity to acid and indicate that M. tuberculosis must acquire sufficient Mg(2+) in order to grow in a mildly acidic environment such as within the phagosome of macrophages.