We have cloned the gene (hgiDIIM) encoding the methyltransferase (MTase) of the SalI isoschizomeric restriction-modification (R-M) system, HgiDII (GTCGAC), into Escherichia coli. The hgiDIIM gene has been isolated from the same plasmid library of Herpetosiphon giganteus strain Hpa2, as was the previously cloned R-M system, HgiDI [AcyI/GRCGYC; Düsterhöft et al., Nucleic Acids Res. 19 (1991) 1049-1056]. Sequencing and functional localization of hgiDIIM revealed an open reading frame (ORF) of 354 codons (39786 Da) with significant homologies to the group of m5C-, rather than the m4C-/m6A-, MTases. Subsequent cloning and analysis of adjacent chromosomal segments led to the identification of two additional ORFs upstream (ORF15, 139 codons) and downstream (ORF68, 611 codons) from hgiDIIM with the same transcriptional orientation as the hgiDIIM gene. However, the expected restriction enzyme function was not found in either of these ORFs.