Chondroitinases are very important tools for the identification and structural analysis of proteoglycans. Enzymic analysis with Flavobacterium heparinum chondroitinases has shown that chondroitin sulphate and dermatan sulphate structures are modified in many human diseases, suggesting a diagnostic value for these enzymes. Furthermore, it was recently shown that F. heparinum chondroitinases AC and B inhibit tumoural cell growth, invasion and angiogenesis. Due to the increasing importance of F. heparinum chondroitinases, we investigated optimized conditions for preparation and assay of chondroitinases AC, B and C. The Dimethylmethylene Blue assay was modified and fully developed to measure the chondroitinase activities of crude extracts of F. heparinum. This method estimates chondroitin sulphate or dermatan sulphate depolymerization upon the digestion of chondroitinase, and was compared with A (232), which measures the unsaturated products formed. Trypticase was the best culture medium, both for bacterial growth and enzyme induction. The chondroitinases were solubilized by ultrasound under conditions that do not completely disrupt the cells, suggesting that they are located at the periplasmic space. Maximum chondroitinase induction occurred in the presence of 0.2-1.0 g/l chondroitin sulphate. Chondroitin sulphate-degradation products were also inducers, but heparin and heparan sulphate were not. Chondroitinases AC, B and C were separated from each other by hydrophobic-interaction chromatography on Phenyl-Sepharose HP. When contaminant proteins were first removed from crude extract by Q-Sepharose, the chondroitinases could be purified to homogeneity in this phenyl-Sepharose chromatographic step.