Asn 212, Arg 243, Tyr 244, Tyr 264, and Lys 274, which are conserved in all known primary sequences of fructose-1, 6-bisphosphatase, are located in the substrate binding domain on the basis of the crystal structure of the enzyme. Mutations of the five residues of porcine liver fructose-1,6-bisphosphatase (Asn212Ala, Arg243-Met, Tyr244Phe, Tyr264Phe, and Lys274Leu) were carried out by site-directed mutagenesis. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism spectrometry. The mutants exhibited kcat values that are similar to those of the wild-type enzyme. The Km values for fructose 1,6-bisphosphate of the mutants are 6- to 44-fold higher than that of the wild-type enzyme. The Ki values for fructose 2,6-bisphosphate and AMP of the mutants increased from 56- to 1950-fold and 12- to 27-fold, respectively, relative to the wild-type enzyme. The alteration of inhibition constants for both inhibitors suggest that these five active site residues are involved in the inhibition by fructose 2,6-bisphosphate and AMP. No apparent differences in secondary structure of the wild-type and mutant forms of fructose-1,6-bisphosphatase were observed as measured by circular dichroism spectrometry. This report demonstrates that Asn 212, Arg 243, Tyr 244, Tyr 264, and Lys 274 not only are the sites for substrate binding, but also play an important role in the binding affinity of inhibitors fructose 2,6-bisphosphate and AMP.