The natural sialidase of Clostridium septicum was purified and characterized in parallel with the recombinant enzyme expressed by Escherichia coli. The two enzymes exhibit almost identical properties. The maximum hydrolytic activity was measured at 37 degrees C in 60 mM sodium acetate buffer, pH 5.3. Glycoproteins like fetuin and saponified bovine submandibular gland mucin, most of them having alpha(2-6) linked sialic acids, are preferred substrates, while sialic acids from gangliosides, sialyllactoses, or the alpha(2-8) linked sialic acid polymer (colominic acid) are hydrolysed at lower rates. alpha(2-3) Linkages are more rapidly hydrolysed than alpha(2-6) bonds of sialyllactoses. The cleavage rate is markedly reduced by O-acetylation of the sialic acid moiety. These properties are similar to those of other secreted clostridial sialidases. The enzyme exists in mono-, di- and trimeric forms, the monomer exhibiting a molecular mass of 125 kDa, which is close to the protein mass of 111 kDa deduced from the nucleotide sequence of the cloned gene.