A series of hydrophilic to hydrophobic surface mutations were prepared at the highly solventexposed lysine 73 of iso-1-cytochrome c to assess the ability of such mutants to affect the energetics of the denatured state. In this report, the aliphatic hydrophobics (leucine, isoleucine, valine, alanine, glycine) were studied. The thermodynamic stability of each of these mutants was determined by guanidine hydrochloride denaturation. Both the free energy of unfolding in the absence of denaturant, ∆G°UH2O, and the slope, m, of a plot of the free energy of unfolding, ∆G°U, versus [guanidine hydrochloride] show significant negative correlations with the 1-octanol to water transfer free energy, ∆Gtr, of the amino acid side chain at position 73. A negative correlation with hydrophobicity is consistent with these mutants leading to more extensive hydrophobic clustering in the denatured state, consistent with the predictions of heteropolymer theory for compact denatured states; an effect operating on the native state energetics should produce a positive correlation of ∆G°UH2O with hydrophobicity. Infrared amide I spectroscopy indicated native state structural perturbations for the glycine 73 and isoleucine 73 mutants. A moderate correlation of ∆G°UH2O was also found with -helix propensity, suggesting that both hydrophobic effects acting on the denatured state and α-helix propensity are affecting the ∆G°UH2O values for these mutants.