The functional reconstitution of Photosystem I reaction center cores by wild-type and mutant PsaC subunits at high ionic strength indicated that hydrophobic interactions are dominant in stabilizing the reaction center. Deletion of the region E27 through C34 in the PsaC backbone resulted in a mutant subunit with impaired reconstitution, confirming that this segment is required for binding. Restoration of the deletion mutant protein to full length using a series of inserts of graded hydrophobicity, including residues with side chains of various sizes. established that this domain is involved in hydrophobic interactions with the reaction center core. The results indicate that a specific proline residue may be necessary to confer the requisite secondary structure in this domain of the PsaC subunit. The overall data provide additional indirect support for orientation of the PsaC subunit on the reaction center such that the Fg cluster is close to the core Fx redox center. We also report that an electrostatic interaction between residue D9 of PsaC and a complimentary basic residue in the core binding site is essential, and most likely ensures the correct orientation of the PsaC subunit on the core during assembly of the reaction center. We previously reported that residue R561 of the PsaB core subunit is also involved in an electrostatic interaction and contributes to the stability of Photosytem I [Rodday, S. M.; Schulz, R.; McIntosh, L.; Biggins, J. Photosynth. Res. 1994, 42185]. We report here that this PsaB core residue is not paired with D9 of the PsaC subunit and postulate that it may be involved in a PsaB(A) intracore interaction.