The proteolytic activity of the extracellular protease of Serratia marcescens was compared with that of trypsin on N, N-dimethyl casein. The peptides produced from exhaustive hydrolysis of alpha casein by the protease and by trypsin were of similar size as measured by gel filtration on P-10 Agarose. We conclude that the protease of S. marcescens in an endopeptidase with trypsin-like activity on proteins, producing oligopeptides. End group analysis of the peptides formed by the S. marcescens protease suggests that the protease has a unique substrate specificity, hydrolyzing only a peptide bond whose carboxyl group is donated by proline. The protease was inactive on the synthetic peptides with proline donating the carboxyl group, but hydrolyzed various types of natural proteins. Its narrow and novel substrate specificity makes this enzyme a potential tool for the determination of the primary structure of proteins.