We have cloned multiple extradiol aromatic ring-cleavage dioxygenase (EDO) genes from a gene library of Pseudomonas aeruginosa JI104, which is a benzene degrader isolated from soil near a gasworks. Southern hybridization analysis revealed that P. aeruginosa JI104 possessed three homologous catechol 2,3-dioxygenase (C230) genes. Nucleotide sequences of the cloned C230 genes, xylE(JI104-1, 2, 3) were almost identical to that of the archetypal C230 gene (xylE(TOL)), which is carried on the TOL plasmid, pWWO. We also cloned another EDO gene, bphC(JI104,) the product of which showed less activity for catechol than did XylE(JI104), but higher activity for 2,3-dihydroxy biphenyl. The nucleotide sequence of bphC(JI104) was identical to that of bphC(KF707) (2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes KF707). The substrate specificities of the four EDOs of P. aeruginosa JI104 mere markedly different from each other. Although XylE(JI104-1) and XylE(TOL) were 94% homologous, the specificities of the gene products for 4-chlorocatechol were extremely different. Results of a study of the chimeric enzymes composed of XylE(JI104-1) and XylE(TOL) N- and C-terminal regions showed that the difference in the specificity for 4-chlorocatechol was dependent on the C-terminal amino acid sequences. All of the isofunctional homologous EDOs in P. aeruginosa JI104 seem to have been derived from a common ancestor and evolved into the present forms in which each EDO is involved in a different degradation pathway and they all coexist in one strain.