BamHI endonuclease and BamHI methylase were used to investigate their specific interaction with the common recognition sequence, GGATCC. Five derivatives of the oligonucleotide, GACGGATCCGTC, containing a variety of single-base analog substitutions within the hexameric recognition core were synthesized. Steady-state kinetics for the reaction of the endonuclease and the methylase showed that both enzymes recognize the sequences by contacting with functional groups exposed in both major and minor grooves of the site but in different ways. Removal or substitution of the 5-methyl group in thymidine blocked the endonuclease reaction completely but still allowed the methylase reaction with less efficiency. The data also showed that the methylase made a critical minor groove contact with the 2-amino group of the first G but the endonuclease did with that of the second G.