Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The ezyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form m-hydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4-hydroxyphthalate were estimated to be 10.5 micrometer and 1.25 mM, respectively, and the Vmax for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5-dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through m-hydroxybenzoate and protocatechuate in P. testosteroni NH1000.