Although multiple chromosomes occur in bacteria, much remains to be learned about their structural and functional interrelationships. To study the structure-function relationships of chromosomes I and II of the facultative photosynthetic bacterium Rhodobacter sphaeroides 2.4.1T, auxotrophic mutants were isolated. Five strains having transposon insertions in chromosome II showed requirements for p-aminobenzoic acid (pABA)-dihydroxybenzoic acid (dHBA), serine, thymine, uracil, or histidine. The His, Thy, and pABA-dHBA mutants reverted to prototrophy at low frequency and concordantly lost their transposon insertions from the genome. The Ser, Ura, and pABA-dHBA mutants were complemented by cosmids that carried the region of chromosome II where the transposon insertions were located. The cosmids used for complementation analysis were selected, on the basis of map position, from a set of overlapping clones that had been ordered by a combination of hybridization and restriction endonuclease mapping. These experiments provide the basis for detailed studies of the structure, function, and interaction between each chromosome, and they demonstrate at this early stage of investigation that no fundamental differences exist between each chromosome.