The incorporation of extensively fluorinated, or fluorous, analogues of hydrophobic amino acids into proteins potentially provides the opportunity to modulate the physicochemical properties of proteins in a predictable manner. On the basis of the properties of small fluorocarbon molecules, extensively fluorinated proteins should be both more thermodynamically stable and self-segregate through "fluorous" interactions between fluorinated amino acids. We have examined the effects of introducing the fluorous leucine analogue l-5,5,5,5',5',5',-hexafluoroleucine (hFLeu) at various positions within the hydrophobic core of a de novo-designed four-alpha-helix bundle protein, alpha(4). The stabilizing effect of hFLeu is strongly dependent on the positions at which it is incorporated, with per-residue DeltaDeltaG(degrees)((fold)) ranging from -0.09 to -0.8 kcal mol(-1) residue(-1). In particular, incorporating hFLeu at all the "a" positions or all the "d" positions of the hydrophobic core, thereby creating an alternating packing arrangement of leucine and hFLeu, leads to very stably folded proteins that exhibit higher per-residue DeltaDeltaG(degrees)((fold)) values than proteins that are packed entirely with hFleu. We conclude that efficient packing of the fluorous amino acid within the hydrophobic core provides a more important contribution to enhancing protein stability than do fluorocarbon-fluorocarbon interactions between fluorinated protein side chains.