Gram-positive Rhodococcus erythropolis strain S1 formed enzymes for the degradation of phthalate when grown in a phthalate-containing minimal medium. The membrane fraction prepared from phthalate-grown cells by ultrasonication converted phthalate to protocatechuate as the final product. Using two membrane-bound enzymes, phthalate 3,4-dioxygenase (PO) and 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH), prepared by solubilization of the membrane fraction, 3,4-dihydroxyphthalate was selectively obtained from phthalata. Fe2+ and Mn2+ stimulated the formation of 3,4-dihydroxyphthalate by the membrane-bound PO and PH system.