A gene cluster encoding cumene (isopropylbenzene)-degrading enzymes was cloned in Escherichia coli JM109 from newly isolated Pseudomonas fluorescens strain IP01. E. coli cells containing pIP103 (10.8-kb insert) converted cumene, other monocyclic aromatic hydrocarbons and biphenyl into the meta-cleaved compounds, whereas the cells containing pIP107D (4.8-kb insert) formed the cis-dihydrodiol compounds, and the cells containing pIP107 (5.2-kb insert) formed the catechol derivatives. We proved that this oxidation system degrades cumene using three enzymes, aromatic-ring dioxygenase, dihydrodiol dehydrogenase, and extradiol dioxygenase. Moreover we determined the nucleotide sequence of a 6,508-bp region encoding aromatic-ring dioxygenase, dihydrodiol dehydrogenase, and extradiol dioxygenase, which subsequently led to identification of six out of seven open reading frames (ORFs) from P. fluorescens IP01. The nucleotide and deduced amino acid sequences for ORF1, 2, 4, and 5, designated cumA1A2A3A4, were found to be similar to those for known multicomponent dioxygenase systems : todC1C2BA from Pseudomonas putida F1 (51-65% amino acid sequence identity) and bphA1A2A3A4 from Pseudomonas pseudoalcaligenes KF707 (59-78%). Two Cys-His pairs in the large subunit of iron-sulfur proteins were conserved in CumA1, and a similar Cys-His pair, conserved in the ferredoxin component of other dioxygenase systems, was found in CumA3. ORF6, designated cumB, was homologous to the dihydrodiol dehydrogenase gene of the fod (58%) and bph (73%) systems, while ORF7, designated cumC, was homologous to todE (47%) and bphC (57%) which encode the meta-cleavage enzymes. Analysis of strain IP01’s nucleotide sequence revealed an overall G+C content of 53% for the cum gene cluster, less than the general G+C content of the chromosome of P. fluorescens.