The dynamic, mitosis-like segregation of bacterial chromosomes and plasmids often involves proteins of the ParA (ATPase) and ParB (DNA-binding protein) families. The conversion of multigenomic aerial hyphae of the mycelial organism Streptomyces coelicolor into chains of unigenomic spores requires the synchronous segregation of multiple chromosomes, providing an unusual context for chromosome segregation. Correct spatial organization of the oriC-proximal region prior to septum formation is achieved by the assembly of ParB into segregation complexes (Jakimowicz et al., 2005; J Bacteriol 187: 3572-3580). Here, we focus on the contribution of ParA to sporulation-associated chromosome segregation. Elimination of ParA strongly affects not only chromosome segregation but also septation. In wild type hyphae about to undergo sporulation, immunostained ParA was observed as a stretched double-helical filament, which accompanies the formation of ParB foci. We show that ParA mediates efficient assembly of ParB complexes in vivo and in vitro, and that ATP binding is crucial for ParA dimerization and interaction with ParB but not for ParA localization in vivo. We suggest that S. coelicolor ParA provides scaffolding for proper distribution of ParB complexes and consequently controls synchronized segregation of several dozens of chromosomes, possibly mediating a segregation and septation checkpoint.