Bacterial actin homologues are thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. They are also thought to control other functions, including cell polarity and chromosome segregation in various organisms. Bacillus subtilis and many other gram-positive bacteria have three actin isoforms, MreB, Mbl, and MreBH, which colocalize in helical structures that span the length of the cell, close to the inner surface of the cytoplasmic membrane. Deletion of the mbl gene has previously been reported to produce viable, although poorly growing, mutant cells. We now show that under normal conditions ⌬mbl cells are nonviable but suppressors allowing growth readily accumulate. In the presence of high concentrations of Mg 2؉ , viable, nonsuppressed mutants can be obtained. A screen for suppressor mutations revealed that deletion of rsgI restores Mg 2؉ -independent growth of the mbl mutant. Recent work has shown that rsgI deletion leads to upregulation of the alternative sigma factor I . The basis of suppression is not yet clear, but it is independent of the Mg 2؉ effect. We found that the construction of a triple mutant lacking all three actin homologues became possible in the rsgI background. Triple mutant cells are spherical, but no significant defect in chromosome segregation was detected.