Hydrophobic interactions play an active role in effective complex formation between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) from Anabaena, where an aromatic amino acid residue on the Fd surface (F65) and three hydrophobic residues (L76, L78, and V136) on the reductase surface have been shown to be essential for the efficient electron transfer (ET) reaction between Fd and FNR (Martínez-Júlvez et al. (2001) J. Biol. Chem. 276, 27498-27510). Since in this system flavodoxin (Fld) can efficiently replace Fd in the overall ET process, we have further investigated if such hydrophobic interactions are also critical in complex stabilization and ET in the FNR/Fld association. Different ET behaviors with Fld are observed for some of the mutations made at L76, L78, and V136 of Anabaena FNR. Thus, the ET interaction with Fld is almost completely lost upon introduction of negatively charged side chains at these positions, while more conservative changes in the hydrophobic patch can influence the rates of ET to and from Fld by altering the binding constants and the midpoint redox potentials of the flavin group. Therefore, our results confirm that nonpolar residues in the region close to the FAD group in FNR participate in the establishment of interactions with Fld, which serve to orient the two flavin groups in a manner such that ET is favored. In an attempt to look for the counterpart region of the Fld surface, the effect produced by the replacement of the only two nonpolar residues on the Fld surface, I59 and I92, by a Lys has also been analyzed. The results obtained suggest that these two hydrophobic residues are not critical in the interaction and ET processes with FNR. The reactivity of these I92 and I59 Fld mutants toward the membrane-anchored photosystem I (PSI) complex was also analyzed by laser flash absorption spectroscopy. From these data, significant effects are evident, especially for the I92 position of Fld, both in the association constant for complex formation and in the electron-transfer rate constant in the PSI/Fld system.