Guanidine-stable chymoelastase (GSC-Elastase), an enzyme isolated from a commercial protease preparation (Pronase), has been shown to be stable and active in the presence of 6.0 M guanidinium chloride (Si egel , S . , et. al . , J . Biol. Chem. 247:4155, J . Pi of . Chem. 248:3233.) The amino acid sequence around the active site serine residue for the protease is similar to that found for bovine chymotrypsin (Awad, W. M. et.al., J. Biol. Chem. 247:4144). This investigation involved the qualitative comparison of the protein cleavage specificity of GSC-Elastase in the presence and absence of denaturant. This specificity was also compared to that found for a-chymotrypsin for the existence of possible similarities in their actionson protein substrates. S-Carboxymethylated lysozyme (CM-HEL) was hydrolyzed in separate trials which were catalyzed by GSC-Elastase in the presence and absence of 6.0 M guanidinium chloride and by a-chymotrypsin. Two-dimensional peptide maps were made from each of the hydrolysates by performing thin-layer chromatography in one direction followed by electrophoresis in a perpendicular direction. The results of the mapping procedure indicated that the cleavage of protein substrates by GSC-Elastase is reproducible and therefore specific under denaturing conditions as well as in the absence of denaturant. These results also suggested that the protein cleavage specificity of GSC-Elastase was found to be significantly different from that of bovine chymotrypsin.