Inhibitors of phenoloxidase were identified in pupae of the housefly, Musca domestica L. The phenoloxidase inhibitors were purified from final instar pupae of the housefly by a combination of ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and reverse-phase high performance liquid chromatography. The potent phenoloxidase inhibitors were heat-stable low molecular weight peptides with an inhibition constant of nM range. To the best of our knowledge, this is the first time that endogenous phenoloxidase inhibitors have been identified among the insects, and probably also among the invertebrates. It is likely that the inhibitors play a central role in regulating the action of active phenoloxidases and will also serve as important tools for understanding the structures and functions of phenoloxidases, as well as their role in insect metamorphosis.