The pKas of 96 acids and bases introduced into buried sites in the staphylococcal nuclease protein (SNase) were calculated using the multiconformation continuum electrostatics (MCCE) program and the results compared with experimental values. The pKas are obtained by Monte Carlo sampling of coupled side chain protonation and position as a function of pH. The dependence of the results on the protein dielectric constant (ϵprot) in the continuum electrostatics analysis and on the Lennard–Jones non-electrostatics parameters was evaluated. The pKas of the introduced residues have a clear dependence on ϵprot, whereas native ionizable residues do not. The native residues have electrostatic interactions with other residues in the protein favoring ionization, which are larger than the desolvation penalty favoring the neutral state. Increasing ϵprot scales both terms, which for these residues leads to small changes in pKa. The introduced residues have a larger desolvation penalty and negligible interactions with residues in the protein. For these residues, changing ϵprot has a large influence on the calculated pKa. An ϵprot of 8–10 and a Lennard–Jones scaling of 0.25 is best here. The X-ray crystal structures of the mutated proteins are found to provide somewhat better results than calculations carried out on mutations made in silico. Initial relaxation of the in silico mutations by Gromacs and extensive side chain rotamer sampling within MCCE can significantly improve the match with experiment. Proteins 2011; © 2011 Wiley-Liss, Inc.