Bothrops asper venom contains a variety of degradative enzymes, including metal-ion dependent proteinases as well as low molecular weight peptides. Two of these peptides, pyroglutamate-glutamine-tryptophan (pEQW) and pyroglutamate-asparagine-tryptophan are present in crude venom at concentrations of about 4.5 and 1 mM, respectively. Proteinase fractions from B. asper are inhibited from digesting oxidized insulin B-chain in vitro by both of these tripeptides with an IC50 for pEQW of approximately 0.3 mM. Digestion of purified myotoxin MIII from B. asper venom is also inhibited in vitro by pEQW, suggesting that similar inhibition of proteinase activities probably occurs in the venom gland. Inhibitory peptides present in venom allow snakes to be protected from their own toxic proteinases and inhibit hydrolysis of venom proteins during storage in the venom gland. Upon dilution, such as when venom is injected into prey, peptide inhibitors dissociate from the proteinase and allow their activation. A simple procedure for isolation of these inhibitory peptides is described.