The enzyme responsible for the degradation of 4-oxalmesaconate was partially purified from Pseudomonas ochraceae grown with phthalate. Column chromatography on DEAE-cellulose caused separation into two distinct enzymes, I and II. 4-Oxalmesaconate was converted into pyruvate and oxalacetate in the presence of MgCl2 and enzymes I and II. Optimum pH of the reaction was observed at pH 8.2 in Tris-HCl buffer. MgCl2 could be replaced by MnCl2 or CoCl2. Both enzymes were stable to heat-treatment at 65 degrees C for 10 min. Analyses of time course, products and substrate specificity of the enzyme reaction accounted for the functions of two enzymes. Enzyme I (molecular weight 55,000, isoelectric point 5.1) hydrated 4-oxalmesaconate to give 4-oxalcitramate and may be classified as a hydrolyase. Enzyme II (160,000, 5.0) catalyzed the aldolitic cleavage of 4-oxalcitramalate to pyruvate and oxalacetate in the presence of MgCl2. Enzyme II also cleaved 4-hydroxy-4-methyl-2-oxoglutarate into pyruvate. Stoichiometry of the enzyme reaction suggested that enzyme II-catalyzed cleavage occurred on only one enantiomer of the substrates. Furthermore, the metabolic pathway for the dissimilation of protocatechuate in P. ochraceae is presented and discussed in comparison with the pathway postulated previously by other workers.