The restriction endonuclease (R.BsuFI) of Bacillus subtilis recognizes the target DNA sequence 5' CCGG. The R.BsuFI gene was found in close proximity to the cognate M.BsuFI gene, which had previously been characterized (1). Cloning of the R.BsuFI gene in E.coli was only possible with the M.BsuFI Mtase gene present on a compatible plasmid. The cloned R.BsuFI gene was expressed in E. coli and restriction activity was observed in vivo and in vitro. The R.BsuFI gene consists of 1185 bp, coding for a protein of 395 amino acids with a calculated molecular weight of 45.6 kD. The R.BsuFI enzyme was purified to homogeneity following overexpression. It presumably works as a dimer and cleaves the 5' CCGG target sequence between the two cytosines to produce sticky ends with 5' CG overhangs, like the isoschizomers R.MspI and R.HpaII. The relatedness between R.BsuFI and R.MspI is reflected by significant similarities of the amino acid sequences of both enzymes. This is the first case where such similarities have been observed between isoschizomeric restriction endonucleases which belong to 5mC specific R/M systems. This observation suggests that R.BsuFI and R.MspI genes derive from a common ancestor. In spite of such functional and evolutionary relatedness, the R/M systems differ in the arrangement of their R and M genes. In the BsuFI system transcription of the two genes is convergent, whereas divergent transcription occurs in the MspI system.