On the basis of the crystal structure of the NAD-dependent cytoplasmic malate dehydrogenase (MDH) and its alignment with NADP-dependent counterparts, the loop region between beta-strand B and alpha-helix C in the dinucleotide-binding fold was predicted as a principal determinant for the coenzyme specificity. Two mutants, EX7 and EX3, of NAD-dependent MDH from Thermus flavus were constructed. In the EX7 mutant, the seven loop amino acids in positions 41-47, Glu-Ile-Pro-Gln-Ala-Met-Lys, were replaced by the corresponding loop residues in the NADP-dependent MDH from chloroplasts, Gly-Ser-Glu-Arg-Ser-Phe-Gln. In the EX3 mutant, Glu-41, Ile-42, and Ala-45 were substituted with the corresponding 3 amino acids in the NADP-dependent chloroplast MDH. In both mutations the coenzyme specificity was altered from NAD to NADP. Especially, the EX7 mutation resulted in a more than 1000-fold improvement in overall catalytic efficiency with NADPH and a 600-fold decrease in the efficiency with NADH as cofactors. Consequently, EX7 mutant was 132 times more efficient with NADPH than NADH without a large decrease in turnover number.