Phthalate-, 4,5-dihydro-4,5-dihydroxyphthalate (DDP)-, 4,5-dihydroxyphthalate (DHP)-, and protocatechuate-accumulating mutants of Pseudomonas testosteroni M4-1 were isolated by transposon insertion mutagenesis. From the DDP-accumulating, transposon-inserted mutant strain M4-122, the 4,5-dihydroxyphthalate decarboxylase gene which is involved in phthalate metabolism was cloned and its nucleotide sequence determined. The structural gene, designated phtD, was 990 bp, corresponding to a protein of 330 amino acid residues (calculated molecular weight 37,200). The phtD gene was expressed in Escherichia coli by its own promoter located upstream of the phtD open reading frame. The promoter shows high homology with the E. coli sigma(70) consensus sequence. The putative amino acid sequence has 77.6% homology with that of the pht5 gene from Pseudomonas putida. The nucleotide sequence of the upstream region of the phtD gene has high homology with that of pht1, the putative positive regulator for the other pht genes of P. putida. The deduced amino acid sequence of the upstream region of the phtD gene also shows high similarity with that of Pht1. From these results, the genes of P. testosteroni and P. putida involved in phthalate metabolism would seem to have evolved from the same origin but diversified to compose different gene orders in different organisms.