Mycobacteria are among the clinically most important pathogens, but still not much is known about the mechanisms of their cell cycle control. Previous studies suggested that the genes encoding ParA and ParB (ATPase and DNA binding protein, respectively, required for active chromosome segregation) may be essential inMycobacterium tuberculosis. Further research has demonstrated that aMycobacterium smegmatis parBdeletion mutant was viable but exhibited a chromosome segregation defect. Here, we address the question if ParA is required for the growth ofM. smegmatis, and which cell cycle processes it affects. Our data show thatparAmay be deleted, but its deletion leads to growth inhibition and severe disturbances of chromosome segregation and septum positioning. Similar defects are also caused by ParA overproduction. EGFP–ParA localizes as pole-associated complexes connected with a patch of fluorescence accompanying two ParB complexes. Observed aberrations in the number and positioning of ParB complexes in theparAdeletion mutant indicate that ParA is required for the proper localization of the ParB complexes. Furthermore, it is shown that ParA colocalizes and interacts with the polar growth determinant Wag31 (DivIVA homologue). Our results demonstrate that mycobacterial ParA mediates chromosome segregation and co-ordinates it with cell division and elongation.