The 4,5-dihydro-4,5-dihydroxyphthalate (DDP)-accumulating Tn5-inserted mutant strain M4-122 of Pseudomonas testosteroni M4-1 was assumed to have a Tn5 insertion at the DDP dehydrogenase gene. We cloned the EcoRI fragment from total DNA of the wild strain corresponding to the Tn5 insertion site of mutant strain M4-122 to verify the existence of the DDP dehydrogenase gene and the arrangement of other genes involved in phthalate metabolism. As a result, the DDP dehydrogenase gene was proven to be located downstream of the 4,5-dihydroxyphthalate decarboxylase gene and the genes were incorporated in a plasmid.