A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 degrees C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K-m and V-max of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml(-1) and 85 mmol min(-1) mg (-1), respectively, and for chondroitin sulfate C, 0.5 mg ml-(1) and 103 mmol min(-1) mg(-1), respectively.