Subtilisin is a bacterial serine protease with a broad specificity in the S1 subsite. It has been very extensively studied using a variety of kinetic and physical techniques. A chemical derivative, thiolsubtilisin, has been subjected to similar studies in order to analyze the effects of the OH to SH conversion on enzyme activity. The native structure of thiolsubtilisin is indicated by a variety of physical techniques. Oligopeptides bind nearly equally well to both enzymes, and a peptide chloromethylketone is much more reactive to thiolsubtilisin than to subtilisin. Both enzymes have a similar level of activity towards activated nonspecific amides and esters. However, thiolsubtilisin is inactive towards highly specific peptide amides and esters. Thiolsubtilisin also does not show good binding to boronic and arsonic acids. The observation that these transition state analog inhibitors bind poorly to thiolsubtilisin while other compounds bind nearly equally well to both enzymes suggests that thiolsubtilisin may not be able to stabilize the transition state during acylation by specific substrates.