Three specific mutants, C54I, C54W, and a double-mutant C54D:C64R of restriction endonuclease BamHI, were generated and studied to investigate the role, if any, of the 54th and 64th cysteine residues in the catalysis of BamHI. The mutation was achieved using the megaprimer approach for PCR. The mutant genes were cloned and characterized by sequencing. The mutant and the wild-type proteins were expressed and purified and their kinetic parameters were determined using short synthetic oligonucleotides as substrates. All mutants had higher K-m values than that of the wild-type enzyme suggesting a decrease in the affinity of the enzyme for its substrate. The mutant protein C54W showed significant changes in the CD spectra vis-a-vis wild-type enzyme and had the lowest K-m/K-cat value among the mutants indicative of changes in the secondary structure of the protein. The melting curves of the mutant proteins overlapped that of the wild-type enzyme. Analysis of the K-cat values in the context of cocrystal structure suggests that the effect of Cys54 mutation is probably through the perturbation of the local structure whereas reduced activity of the double mutant is consistent with the substrate-assisted catalysis mechanism.