Heparinases I, II and III from F. heparinum cleave heparin-like molecules, with a high degree of substrate specificity, at the glucosamine-uronate linkage by elimination, leaving an unsaturated C4-C5 bond in the uronic acid. The primary sequences of these enzymes have been reported earlier. In this study we perform a comparative analysis of the properties and primary sequences of heparinase I, II and III. Alignment of the primary sequences revealed little sequence homology (15% residue identity in a LALIGN alignment) at both DNA and amino acid levels. There are three basic clusters in heparinase II satisfying the heparin binding consensus sequence with one of the sequences sharing homology with a consensus sequence in the heparin binding site of heparinase I and two basic clusters in heparinase III. Similar to heparinase I, there are two putative 'EF-hand' calcium coordinating motifs in heparinase III, while heparinase II does not contain any such motifs. Recombinant heparinases II and III's degradation of the substrate and the subsequent separation of the oligosaccharide products by POROS anion exchange chromatography were identical to those obtained from native heparinases II and III from F. heparinum.