One free -SH group in the heavy chain of alpha-clostripain reacts rapidly with N-tosyllysine chloromethyl ketone which inactivates the enzyme. Iodoacetic acid also reacts with the thiol group required for enzyme activity but more slowly. A tryptic peptide containing the reactive sulfhydryl group labelled with iodo[1-14C]acetic acid was isolated and determined to be Gln-Ser-Val-Asp-Leu-Leu-Ala-Phe-Asp-Ala-Cys-Met. All other cysteine peptides were isolated from the trypsin hydrolysate of the [14C]carboxymethylated enzyme. Moreover N-terminal and C-terminal sequences of both chains of alpha-clostripain were determined. The sequences representing 20% of the primary structure of alpha-clostripain are not homologous with either other cysteine proteinases or with any other protein structure known to date.