The specific methylation site for Alu I methylase was the cytosine nucleotide in Alu I sequence. The position of the methylated cytosine nucleotide was determined by the chemical cleavage reactions of the Maxam-Gilbert DNA sequencing procedure. As expected, the methylated cytosine nucleotide bands were disappeared on C+T and C lanes on 1296 sequencing gels. Alu I methylase was maximally active at near pH 7.5 in the presence of 50 mM NaCI. The methylase did not require Mg^(++) for activity, and obeyed Michaelis-Menten Kinetics with respect to both AdoMet and DNA. At 37℃, the Km for AdoMet was 0.44 μM, that for the Alu I site of pBR 322 DNA was 4.03 nM, and the corresponding turnover numbers were 1.83 methyl transfer per minute per monomer and 1.61 transfers per minute per monomer, respectively.