To understand the functional groups required for sequence recognition, dodecanucleotides containing ClaI sequence in the middle positions with modified bases were synthesized and used as substrates for ClaI endonuclease and ClaI methylase reactions. For the modification, dU, 5-bromo-dU, and dI were used. Km values of the two enzymes were determined for normal and modified oligonucleotides. Our data showed that 5-CH3 groups of the first and second T residues of the hexanucleotide sequence were essential contact points for ClaI methylase. However, ClaI endonuclease was still active without either one of those methyl groups with elevated Km values. Bromine could compensate significantly for the loss of 5-CH3 groups at both position. On the other hand, the 2-amino group of G residue appeared to be an essential contact point for both enzymes. It has been concluded that ClaI endonuclease and ClaI methylase recognize the sequence in different ways.