Arg-38 is an internal residue of mitochondrial cytochrome c that is close to heme propionate-7. Previous work comparing the behavior of cytochromes c from several species [Moore, G. R., Harris, D. E., Leitch, F. A., & Pettigrew, G. W. (1984) Biochim. Biophys. Acta 764, 331-342] has suggested that Arg-38 lowers the pKa of this propionate group and thereby accounts for the relative pH independence of the cytochrome c reduction potential from pH 5 to pH 8. The influence of Arg-38 on the oxidation-reduction equilibrium of yeast iso-1-cytochrome c has now been investigated by electrochemical, NMR, and theoretical analysis of six specifically mutated forms of this protein in which Arg has been replaced by Lys, His, Gln, Asn, Leu, or Ala. As the electron-withdrawing character of the residue at position 38 decreases, the reduction potential of the protein also decreases, with the largest decrease (ca. 50 mV) observed for the Ala variant. However, the variation in the reduction potentials of the mutants as a function of pH was similar to that observed for the wild-type protein. The effects of some of these mutations on the pKa values of His-33 and His-39 have been determined by NMR spectroscopy and found to be minimal. Calculations of the electrostatic free energy for the Leu-38 variant predict a decrease in the reduction potential of this mutant that is remarkably close to that observed experimentally. This work establishes that while Arg-38 contributes to the relatively high reduction potential of cytochrome c, this residue does not appear to be the sole functionality responsible for lowering the heme propionate-7 pKa.