Bacitracin is commonly used in metabolic studies as an insulinase inhibitor. The many isoforms of the commercial preparation were fractionated by charge and size in order to find the most active rat-muscle insulinase inhibitors. CM-Sepharose chromatography revealed that most of the inhibitory activity was contained in a fraction (CM-Inh) that amounted to less than 5% of the mixture. The CM-Inh fraction could be further separated by size on Bio-Gel P4 columns. Six subgroups, each with characteristic specific activity, were isolated. The most potent inhibitor fractions have no antibiotic activity and have molecular weights about twice that of bacitracin A. The peaks isolated by means of Bio-Gel P4 chromatography can be further fractionated by reversed phase HPLC on a C8 column, and by electrophoresis on nonreducing acrylamide gels.