Abstract: In accordance with the present invention, there is provided a novel restriction endonuclease and its DNA obtainable from Helicobacter pylori CH4 (NEB#1236), hereinafter referred to as “HpyCH4III”, which endonuclease: (1) recognizes the nucleotide sequence 5′-ACNGT-3′ in a double-stranded DNA molecule as shown below, 5′-ACN↓GT-3′ 3′-TG↑NCA-5′  (wherein G represents guanine, C represents cytosine, A represents adenine, T represents thymine and N represents either G, C, A, or T); (2) cleaves said sequence in the phosphodiester bonds between the N and G as indicated with the arrows; and (3) cleaves double-stranded PhiX174 DNA to produce 15 fragments, including fragments of 1284, 814, 536, 517, 454, 404, 302, 292, 270 and 222 base pairs, and 5 fragments smaller than 200 base pairs.