Stabilization of protein structures and protein-protein interactions are critical in the engineering of industrially useful enzymes and in the design of pharmaceutically valuable ligands. Hydrophobic interactions involving phenylalanine residues play crucial roles in protein stability and protein-protein/peptide interactions. To establish an effective method to explore the hydrophobic environments of phenylalanine residues, we present a strategy that uses pentafluorophenylalanine (F5Phe) and cyclohexylalanine (Cha). In this study, substitution of F5Phe or Cha for three Phe residues at positions 328, 338, and 341 in the tetramerization domain of the tumor suppressor protein p53 was performed. These residues are located at the interfaces of p53-p53 interactions and are important in the stabilization of the tetrameric structure. The stability of the p53 tetrameric structure did not change significantly when F5Phe-containing peptides at positions Phe328 or Phe338 were used. In contrast, the substitution of Cha for Phe341 in the hydrophobic core enhanced the stability of the tetrameric structure with a T(m) value of 100 degrees C. Phe328 and Phe338 interact with each other through pi-interactions, whereas Phe341 is buried in the surrounding alkyl side-chains of the hydrophobic core of the p53 tetramerization domain. Furthermore, high pressure-assisted denaturation analysis indicated improvement in the occupancy of the hydrophobic core. Considerable stabilization of the p53 tetramer was achieved by filling the identified cavity in the hydrophobic core of the p53 tetramer. The results indicate the status of the Phe residues, indicating that the "pair substitution" of Cha and F5Phe is highly suitable for probing the environments of Phe residues.