IrelandThe nucleotide sequence of the chromosomally encoded type II ScrFI restriction/modif ication system from Lactococcus lactis subsp. cremoris UC503 was completed. The ScrFl restriction endonuclease (ENase) has previously been shown to specifically recognize 5' CCNGG 3' sites, cleaving after the second cytosine and the degenerate central base. The ENase gene (scrFIR; 862 bp) was located between, and co-directionally transcribed with, two formerly characterized 5-methylcytosine methyltransferase genes, which encode proteins that independently confer protection against Scrfl digestion. scrFlR codes for a protein of 272 amino acids with a predicted molecular mass of 31 470 Da, which agrees favourably with a previously estimated molecular mass of 34 kDa for this enzyme. The deduced sequence of this protein did not show any significant homology with known protein sequences, including the isoschizomeric Ssoll ENase from Shigeh sonnei. The ENase gene was cloned and expressed in Escherichia coli and Lactococcus; however, no in wiwo restriction of phage was observed, suggesting that expression of the ENase gene may be repressed, or that the appropriate expression signals may be absent in the cloned constructs. The ability of ScrFl to cleave non-canonically modified 5' CCNGG 3' sequences suggested that some ScrFI sites may require complex modifications to fully impair digestion by this enzyme.