The role of Mg ions in the hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction have been studied using accurate values of proton and Mg stability constants of phosphoribosylpyrophosphate (P-Rib-PP) determined from pH titration data. The results obtained favor the conclusion that the dimagnesium salt of P-Rib-PP is the true substrate of the enzyme. The other specoiefs P -Rib-PP do not appreciably affect the initial reaction rate. The inhibition of the hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction observed at highM gClz concentration can be attributed to a competitive inhibition of M&' with respect to the dimagnesium salt of P-Rib-PP, suggesting that these ionic specbiiensd to the same enzyme form. At a fixed [P-Rib-PP,,,], the concentration of its dimagnesium complex is a sigmoidal function of M&Iz concentration, suggesting that caution must be employed in the interpretation of sigmoidal saturation curves for P-Rib-PP-utilizing enzymes when low and not constant concentrations of the divalent cation are used