In this study, we cloned and sequenced a DNA fragment from an ordered cosmid library of Helicobacter pylori NCTC 11638 which confers to a siderophore synthesis mutant of Escherichia coli (EB53 aroB hemA) the ability to grow on iron-restrictive media and to reduce ferric iron. Sequence analysis of the DNA fragment revealed the presence of an open reading frame with high homology to the ribA gene of Bacillus subtilis. This gene encodes a bifunctional enzyme with the activities of both 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase and GTP cyclohydrolase II, which catalyze two essential steps in riboflavin biosynthesis. Expression of the gene (designated ribBA) resulted in the formation of one translational product, which was able to complement both the ribA and the ribB mutation in E. coli. Expression of ribBA was iron regulated, as was suggested by the presence of a putative FUR box in its promotor region and as shown by RNA dot blot analysis. Furthermore, we showed that production of riboflavin in H. pylori cells is iron regulated. E. coli EB53 containing the plasmid with H. pylori ribBA excreted riboflavin in the culture medium, and this riboflavin excretion also appeared to be iron regulated. We postulate that the iron-regulated production of riboflavin and ferric-iron-reduction activity by E. coli EB53 transformed with the H. pylori ribBA gene is responsible for the survival of EB53 on iron-restrictive medium. Because disruption of ribBA in H. pylori eliminates its ferric-iron-reduction activity, we conclude that ribBA has an important role in ferric-iron reduction and iron acquisition by H. pylori.