Under iron limitation, the plant pathogen E. chrysanthemi produces the catechol-type siderophore chrysobactin that acts as a virulence factor. It can also use enterobactin as a xenosiderophore. We began this work by sequencing the 5' upstream region of the fct cbsCEBA operon which encodes the ferric chrysobactin receptor and proteins involved in synthesis of the catechol moiety. We identified a new iron-regulated gene, cbsH, transcribed divergently relative to the fct gene, the translated sequence of which is 45.6 % identical to that of the E. coli ferric enterobactin esterase. Insertions within this gene interrupt the chrysobactin biosynthetic pathway by exerting a polar effect on a downstream gene with some sequence identity to the E. coli enterobactin synthase gene. These mutations had no effect on the ability of the bacterium to obtain iron from enterobactin, showing that a functional cbsH gene is not required for iron removal from ferric enterobactin in E. chrysanthemi. The cbsH-negative mutants were less able to utilise ferric chrysobactin and this effect was not caused by a defect in the transport per se. In a non-polar cbsH-negative mutant, chrysobactin accumulated intracellularly. These defects were rescued by the cbsH gene supplied on a plasmid. The amino acid sequence of the CbsH protein revealed characteristics of the S9 prolyl oligopeptidase family. Ferric chrysobactin hydrolysis was detected in cell extracts from a cbsH-positive strain, that was inhibited by diisopropyl fluorophosphate. These data are consistent with the fact that chrysobactin is a D-lysyl-L-serine derivative. Mössbauer spectroscopy of whole cells at various states of 57Fe chrysobactin uptake showed that this enzyme is not required for iron removal from chrysobactin in vivo. The CbsH protein may therefore be regarded as a peptidase preventing the bacterial cells to be intracellularly iron-depleted by chrysobactin.