Chondroitin sulfates are the glycosaminoglycan chains of proteoglycans critical in the normal development and pathophysiology of all animals. Chondroitinase ACII, a polysaccharide lyase originally isolated from Arthrobacter aurescens IAM 110 65, which is widely used in the analysis and study of chondroitin structure, is no longer commercially available. The aim of the current study was to prepare recombinant versions of this critical enzyme for the glycobiology research community. Two versions of recombinant chondroitinase ACII were prepared in Escherichia coli, and their activity, stability, specificity and action pattern were examined, along with a non-recombinant version secreted by an Arthrobacter strain. The recombinant enzymes were similar to the enzyme obtained from Arthrobacter for all examined properties, except for some subtle specificity differences towards uncommon chondroitin sulfate substrates. These differences are believed to be due to either post-translational modification of the Arthrobacter secreted enzyme or other subtle structural differences between the recombinant and natural enzymes. The secreted chondroitinase can serve as a suitable replacement for the original enzyme that is currently unavailable, while the recombinant ones can be applied generally in the structural determination of most standard chondroitin sulfates.