Xanthomonadins are yellow, membrane-bound pigments produced by members of the genus Xanthomonas. We identified an ethyl methanesulfonate-induced Xanthomonas oryzae pv. oryzae mutant (BXO65) that is deficient for xanthomonadin production and virulence on rice, as well as auxotrophic for aromatic amino acids (Pig Vir Aro). Reversion analysis indicated that these multiple phenotypes are due to a single mutation. A genomic library of the wild-type strain was used to isolate a 7.0-kb clone that complements BXO65. By transposon mutagenesis, marker exchange, sequence analysis, and subcloning, the complementing activity was localized to a 849-bp open reading frame (ORF). This ORF is homologous to the aroE gene, which encodes shikimate dehydrogenase in various bacterial species. Shikimate dehydrogenase activity was present in the wild-type strain and the mutant with the complementing clone, whereas no activity was found in BXO65. This clone also complemented an Escherichia coli aroE mutant for prototrophy, indicating that aroE is functionally conserved in X. oryzae pv. oryzae and E. coli. The nucleotide sequence of the 2.9-kb region containing aroE revealed that a putative DNA helicase gene is located adjacent to aroE. Our results indicate that aroE is required for normal levels of virulence and xanthomonadin production in X. oryzae pv. oryzae.