The biosynthesis of the aryl carotenoid, chlorobactene, was examined in the green sulfur bacterium, Chlorobium limicola f. thiosulfatophilum. Nicotine, which was used to inhibit carotenoid cyclization, caused the accumulation of the acyclic carotenoid, lycopene. Cells reincubated in fresh medium, after removal of nicotine, synthesized chlorobactene more readily from newly synthesized lycopene rather than from the pool of lycopene accumulated during nicotine inhibition. When the cells were reincubated in the presence of diphenylamine, which inhibited de novo carotenogenesis, a portion of the lycopene which had accumulated during nicotine inhibition was converted into chlorobactene. There was no evidence that neurosporene, rather than lycopene, was the precyclization intermediate. The involvement of gamma-carotene as the cyclic precursor of chlorobactene also was shown. The pathway for chlorobactene biosynthesis is discussed in terms of a possible arrangement of the enzymes involved in carotenoid biosynthesis.