Previous studies have demonstrated that His 12 plays a major role in the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A). Here, amino acid replacement experiments show that His 12+ stabilizes the C-peptide helix chiefly by interacting with Phe 8. The Phe 8 ... His 12+ ring interaction is specific for the protonated form of His 12 (His 12+) and the interaction is not screened significantly by NaCl, unlike the charged group ... helix dipole interactions studied earlier in C-peptide. Analogs of C-peptide that are unable to form the Phe 8 ... His 12+ interaction show large increases in helix content for Phe----Ala and His----Ala. Therefore, the helical tendencies of the individual residues Phe, His, and Ala are important in determining the result of a replacement experiment. Since the side chains of Phe 8 and His 12 probably interact within the N-terminal helix of ribonuclease A, the existence of the Phe 8 ... His 12+ interaction in the isolated C-peptide helix adds to the evidence that the C-peptide helix is an autonomous folding unit.