Bacitracin, as purchased from biochemical supply companies, is a mixture of more than 30 different substances. The major antibiotic isoforms A and B account for about 60% of the mixture. A newly identified impurity in some, but not all, of the bacitracin lots is a powerful subtilisin-type protease capable of cleaving many proteins including protein disulfide isomerase (PDI), myosin, and a variety of artificial substrates Thus, it is important for investigators who use bacitracin as a protease or other enzyme inhibitor to determine if the bacitracin they are using is contaminated with a protease enzyme. If it is present, they may have to reinterpret their results and retest with an enzyme-free bacitracin reagent.