Sequence perturbation analysis is a powerful method to reveal roles of an amino acid residue in function and stability of a protein. By using and improving this method, we studied roles of highly conserved Arg44 of Escherichia coli dihydrofolate reductase (DHFR) in its function and stability. Here, we introduced systematic amino acid substitutions at this position and found that all 19 kinds of amino acid substitutions were tolerated, but the mutations significantly reduced the enzymatic activity and the binding affinity toward the cofactor NADPH. Moreover, the mutational effects on the cofactor binding affinity were well correlated with those on the catalytic activity, indicating that the R44X mutations affect the catalytic activity mainly by modulating the cofactor binding affinity. On the other hand, thermal denaturation measurements showed that most mutations stabilized the protein. Comparison between the mutational effects and various amino acid indices taken from the AAindex database indicated that hydrophobicity and polarity are key determinants of amino acids favorable at this position. These results suggest that through electrostatic interactions Arg44 plays a functional role in retaining the cofactor binding affinity at the cost of the protein stability.