The photosynthetic bacterium Rhodobacter sphaeroides accumulates 5-aminolevulinic acid (ALA), which is a precursor in tetrapyrrole biosynthesis, under light illumination and upon addition of levulinic acid as an inhibitor of ALA dehydratase. To generate an industrial strain which produces ALA in the absence of light, we sequentially mutated R. sphaeroides CR-286 using N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The mutant strains were screened by cultivating in the absence of light and assayed for ALA by the Ehrlich reaction in a 96-well microtiter plate. The mutant strain CR-386, derived from R. sphaeroides CR-286, was selected as a mutant that exhibited significant ALA accumulation. While CR-286 required light illumination for ALA production, CR-386 was able to accumulate 1.5 mM ALA in the presence of 50 mM glucose, 60 mM glycine, 15 mM levulinic acid and 1.0% (w/v) yeast extract under conditions of agitation in the absence of light. The mutant strain CR-450, derived from strain CR-386, was selected further as a mutant that exhibited significant ALA accumulation but no accumulation of aminoacetone, analogue of ALA. CR-450 accumulated 3.8 mM ALA under the same conditions. In the presence of 50 mM glucose, 60 mM glycine, 5 mM levulinic acid and 1.0% (w/v) yeast extract, the mutant strain CR-520, derived from strain CR-450, and strain CR-606, derived from strain CR-520, accumulated 8.1 mM and 11.2 mM ALA, respectively. In batch fermentation, the strain CR-606 accumulated 20 mM ALA over 18 h after the addition of glycine, levulinic acid, glucose and yeast extract.