To elucidate the role of flexible loops in the stability and function of Escherichia coli dihydrofolate reductase (DHFR), glycine-67, glycine-121, and alanine-145 in the three different loops were substituted with several amino acids by site-directed mutagenesis. The free energy change of unfolding by urea decreased with an increase in the hydrophobicity and volume of amino acids introduced at any site. In the enzyme reaction, Km was only slightly influenced, but kcat was decreased by mutation, especially with significant effects at site 121. Adiabatic compressibility also largely changed by mutation, indicating the modified volume fluctuation of the native state. Further, nonadditive effects were observed in the stability and function of the double mutants at sites 67 and 121 although they are separated by 27.4 A. These results indicate that the effects of mutations extend to almost the whole protein molecule via reconstituted atomic packing or long-range interaction, and then the loop regions play important roles in the stability, fluctuation, and function of this enzyme.