Chlorosis is one of the symptoms of bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria, which induces chlorosis before any other symptoms appear on tomato. We report characterization of a 2.1-kb gene called early chlorosis factor (ecf). The gene ecf encodes a hydrophobic protein with similarity to four other proteins in plant pathogens, including HolPsyAE, and uncharacterized gene products from X. campestris pv. campestris and X. axonopodis pv. citri, and, at the tertiary structure level, to colicin Ia from Escherichia coli. We demonstrate that the associated phenotype is hrp dependent, and that the ecf gene product appears to be translocated to host cells. The gene ecf has no impact on electrolyte leakage or on bacterial growth in planta in response to infection. Concentrated culture filtrates do not produce chlorosis. Study of its role in Xanthomonas spp.-tomato interactions will forward our understanding of symptom production by plant pathogens and allows further investigation into the mechanisms of bacterial virulence and production of symptoms.