In this study, the substrate binding sites and mode of binding of benzhydroxamic acid (BHA) in the low-spin cyanide-ligated form of horseradish peroxidase isoenzyme C (HRP-C) have been identified and characterized using the X-ray crystallographic structure of HRP-C in the substrate-free form, in combination with the programs AUTODOCK and AMBER. Two criteria were used to select the most favorable binding site : the interaction energy of BHA with the protein and the mobility of BHA in each binding site. Using these criteria, the binding site located on the distal side of the heme and surrounded by His42, Arg38, Pro139, Leu138, Ala140, Phe68, Pro141, Gly69, Phe179, Phe41, Asn70, and Ser73 was found most promising. Computed distances between atoms in BHA and atoms in residues of HRP-C/CN were, in general, in good agreement with a subset of corresponding distances derived from H-1 NMR data. In addition, two strong H bonds of BHA, with Arg38 and the N atom of the cyanide ligand, and two polar interactions of BHA, with His42 and Pro139, were found, consistent with the relatively high binding affinity of BHA for HRP-C/CN. The second most favorable binding site identified was located toward the proximal side of the heme at a distance of only 10 Angstrom from the first binding site, as described above, suggesting it as a temporary storage place for the radical produced by oxidation of the first substrate molecule. This small displacement would allow accommodation of a second substrate molecule in the first site and dimerization to occur after the second substrate radical is formed through a second one-electron oxidation step. Although it is not known whether BHA forms dimers due to oxidation by HRP-C, other phenolic substrates that do form dimers may occupy both the primary binding site, where oxidation occurs, and the radical holding site, to facilitate dimer formation.