Long-range coulombic interaction energies between surface-charges in barnase and subtilisin have been determined to provide data for calibrating theoretical methods. The pKa of His18 in barnase can be measured accurately by titrating the fluorescence of Trp94 that is significantly quenched on protonation of His18. The pKa of His64, the active site base of subtilisin, has previously been shown to be measured accurately from the pH dependence of kcat /Km for the hydrolysis of substrates. The titration curves of both histidine residues fit the theoretical equations for the ionization of single groups with great precision; the Hill constants for wild-type and mutant enzymes are all close to 1·0. The coulombic interaction energies of distant charged side-chains with the protonated form of His18 and His64 have been measured from changes in pKa of these residues on mutation of those charged side-chains. The interaction energies between single charges on the surfaces of the proteins at low ionic strength are small, some 0·3-0·5 kcal mol-1 at a distance of 12 Å, and fall gradually with distance to 0·05-0·3 kcal mol-1 at 20 Å. Multiple mutations are frequently additive. Effects are larger in subtilisin than in barnase, possibly related to the degree of solvent exposure of the charge. These data have been used to benchmark the finite-difference method of calculating electrostatic interactions as implemented in the program DelPhi. There is reasonable agreement between the calculated and measured results as a function of both position and ionic strength.