Porcine NADH-cytochrome b5 reductase catalytic domain (Pb5R) has the RXY(T/S)+(T/S) flavin-binding motif that is highly conserved among the structurally related family of flavoprotein reductases. Mutations were introduced that alter the Arg63, Tyr63, and Ser99 residues within this motif. The mutation of Tyr63 to either alanine or phenylalanine destabilized the protein, produced an accelerated release of FAD in the presence of 1.5 M guanidine hydrochloride, and decreased the cat values of the enzyme. These results indicate that Tyr63 contributes to the stability of the protein and is important in the electron transfer from NADH to FAD. The mutation of Ser99 to either alanine or valine, and of Arg63 to either alanine or glutamine increased both the Km values for NADH (KmNADH) and the dissociation constant for NAD+ (KdNAD+). However, the mutation of Ser99 to threo-nine and of Arg63 to lysine had very little effect on the KmNADH and KdNAD+ values, and resulted in small changes in the absorption and circular dichroism spectra. These results suggest that the hydroxyl group of Ser99 and the positive charge of Arg63 contribute to the maintenance of the properties of FAD and to the effective binding of Pb5R to both NADH and NAD+. In addition, the mutation of Arg68 to either alanine or glutamine increased the apparent Km values for porcine cytochrome b5 (Pb5), while changing Arg63 to lysine did not. The positive charge of Arg63 may regulate the electron transfer through the electrostatic interaction with Pb5. These results substantiate the important role of the flavin-binding motif in Pb5R.