The nucleotide sequence of a 2837-base pairs (bp) EcoRI-PvuI fragment of Bacillus stearothermophilus LV chromosomal DNA encoding the bstLVIM gene was determined. It revealed a large open reading frame (ORF) of 1737 bp specifying a methylase of 579 amino acid (aa) residues and Mr 66,831. This was in agreement with the size estimated for the M.BstLVI (similar to 67 kDa) purified from Escherichia coli cells harboring a recombinant plasmid containing the bstLVIM gene and with results of transcription-translation experiments performed in vitro. Upstream the bstLVIM gene and in the opposite transcriptional orientation, there is a 81-aa ORF that showed great homology with the regulatory C proteins identified in other type II restriction and modification (R-M) systems. This 81-aa ORF precedes a truncated ORF of 86 aa which in turn may represent the structural gene for the BstLVI restriction endonuclease.