An endonuclease was purified from the digestive juice of silkworm larvae, Bombyx mori. The enzyme hydrolyses both DNA and RNA endolytically with little base specificity, yielding di- and trinucleotides terminating in 5'-phosphate. Polynucleotides terminated in 3' -phosphate gave dinucleosidemonophosphates and trinucleosidediphosphates from 5'-end, di- and trinucleotides from inside, and mononucleosidediphosphates, dinucleosidetriphosphates, and trinucleosidetetraphosphates from 3'-end, respectively. Taking advantage of these peculiar nature of the nuclease, an oligonucleotide addition procedure was devised to sequence nucleic acids from the overlapping pattern of the fragments of digestion by the nuclease. Proven and potential usefulness of the silkworm endonuclease in the structural work on nucleic acids and specificity analysis of nucleolytic enzymes were discussed.