A restriction endonuclases (ENase) designated EcoO44I was purified without non specific nucleases from enteropathogenic Escherichia coli O44 Hiromi strain of affected human origin. The yield was 1, 100 units/g of wet cells. The EcoO44I ENase recognized and cleaved the specific sequence of 5'-GGTCTC-3' (1/5) as was the case with Eco31I or BsaI ENase. Because of the stability and high yield, EcoO44I would be useful for recombinant DNA technology after isolation of EcoO44-positive, avirulent mutant strains of E. coli O44 Hiromi.