Riboflavin-requiring mutants were isolated from Corynebacterium ammoniagenes. One of the mutants, RK122, accumulated 6,7-dimethyl-8-ribityllumazine (DMRL), a direct precursor of riboflavin, in the culture medium. Chromosomal DNA fragments that complement the riboflavin requirement of RK122 were cloned and sequenced. Sequence analysis revealed that the structure of the riboflavin biosynthetic genes of C. ammoniagenes showed significant similarity to those of Bacillus subtilis.