The XcyI restriction-modification system from Xanthomonas cyanopsidis recognizes the sequence, CCCGGG. The XcyI endonuclease and methylase genes have been cloned and sequenced and were found to be aligned in a head to tail orientation with the methylase preceding and overlapping the endonuclease by one base pair. The nucleotide sequence codes for an N4 cytosine methyltransferase with a predicted molecular weight of 33,500 and an endonuclease comprised of 333 codons and a molecular weight of 36,600. Sequence comparisons revealed significant similarity between the XcyI, CfrI and SmaI methylisomers. In contrast, no similarity was detected between the primary structures of the XcyI and SmaI endonucleases. The XcyI restriction-modification system is highly homologous to the XmaI genes, although the DNA sequences flanking the genes rapidly diverge. The sequence of the XcyI endonuclease contains two motifs which have recently been identified as essential to the activity of the EcoRV endonuclease.