β-Citryl-l-glutamate-hydrolysing enzyme (β-CGHE) was purified from rat testis particulate fraction 13 000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, β-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed β-citryl-l-glutamate (β-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the β-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. β-CGHE was also potently inhibited by an excitatory amino acid agonist, l-quisqualate, but not by another agonists,N -methyl-d-aspartate and kinate. It had high substrate specificity for β-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.