Chlorocatechol 1,2-dioxygenase from Rhodococcus erythropolis 1CP was purified to homogeneity. In contrast to chlorocatechol 1,2-dioxygenase from Gram-negative strains which have a very broad substrate tolerance, the Rhodococcus enzyme was relatively more specific and had a distinct preference for 4-substituted catechols. Protein and metal analysis indicate an unusual stoichiometry of one atom each of iron and manganese/64-kDa homodimer. The N-terminal amino acid sequence (27 residues) of the Rhodococcus chlorocatechol 1,2-dioxygenase was determined and exhibited 15-22% identity to the published sequences of catechol 1,2-dioxygenases and other chlorocatechol 1,2-dioxygenases. Antiserum was raised in rabbits and antibodies against Rhodococcus chlorocatechol 1,2-dioxygenase were affinity purified. Dot-blot analysis revealed a very weak reaction between the antibodies and partially purified chlorocatechol 1,2-dioxygenases from Alcaligenes eutrophus JMP134 and Pseudomonas putida 87. No reaction between these antibodies and above enzymes was observed using Western blotting. Kinetic and immunochemical data as well as comparison of subunit molecular mass and suggest that the Rhodococcus enzyme differs significantly from the known highly similar chlorocatechol 1,2-dioxygenases of Gram-negative strains and seems to be only distantly related to them.