Mutants of Salmonella typhimurium lacking protease II, an endoprotease with trypsin-like specificity, have been isolated. These mutants can be identified by using the chromogenic substrate N-methyl-N-p-toluenesulfonyl-L-lysine beta-naphthyl ester to screen colonies growing on agar for the presence of the enzyme. All of the mutations isolated map at locus tlp (typsin-like protease) which is cotransducible (approximately 1%) using phage P1 with tre (trehalose utilization) at approximately 58 min on the Salmonella map. Double mutants lacking both protease I and protease II have been constructed. These strains grew normally. They were able to degrade abnormal proteins and to carry out protein turnover during carbon starvation at the same rate as the wild type.