Hypoxanthine phosphoribosyltransferases (HPRT; EC 2.4.2.8) catalyze the transfer of a phosphoribosyl group from 5-phospho-a-D-ribosyl-1-pyrophosphate (PRPP) to a purine base (hypoxanthine, guanine or xanthine) to form pyrophosphate (PPi) and a purine nucleotide, inosine monophosphate (IMP), guanosine monophosphate (GMP), or xanthosine monophosphate (XMP), respectively. The active site of these enzymes is defined, in large part, by four loops. Residue Arginine 99 is located at the base of active site loop II allowing interactions with residues from loop II of the neighboring monomer or with the substrates PRPP or PPi. We have carried out a site-directed mutagenesis study in order to understand the role of this residue on the enzymatic activity and binding of the substrates by the protein. All mutants obtained (glycine, histidine, serine, phenylalanine, leucine, valine and isoleucine) showed small effects on the catalytic constant, kcat for all the substrates of the forward (PRPP and hypoxanthine) and the reverse (IMP and PPi) reactions. A strong effect was observed for the Km for all the substrates, with the small the side chain of the mutated residue the biggest the effect on Km. We have also studied the effect of PRPP on the thermal stability of HPRT. Small amounts (1x Km) of this substrate increases the Tm of the wild type enzyme by 6°C, and saturating concentrations (5x Km) did not increase further its thermal stability. In the case of the mutants, saturating concentrations increased the T_m between 15 and 18°C. These results show a role of this residue (Arg99) on the binding of substrates either through a direct interaction or by an indirect modulation through the interaction of residues of loop II.