NAD + -dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP + but still retained substantial activity with NAD + . Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD + and NADP + with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K m for NADP + than that of the wild type enzyme for NAD + was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K m values for PGE 2 of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP + but not with NAD + . The triple mutants showed a decrease in K m for NADP + but an increase in K m for PGE 2 . Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K m values for both NADP + and PGE 2 to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD + to NADP + by changing a few critical residues near the N-terminal end.