A new type-II restriction endonuclease SphI, has been partially purified from Streptomyces phaeochromogenes. SphI recognizes the hexanucleotide sequence 5'-GCATGC and cleaves it at the position marked by the arrow. This nucleotide sequence is present twice in SV40 DNA, four times lambda DNA and only once in the cloning vehicles pBR322, pBR325, pBR327 and pBR328.