alpha-Helical polypeptides carrying L-4-biphenylalanine and L-1-naphthylalanine that are separated by up to 11 amino acid units or 19.5 Angstrom were prepared by stepwise peptide synthesis. Singlet energy transfer from the biphenyl group to the naphthyl group was investigated with steady-state fluorescence spectroscopy, and the efficiency of energy transfer was evaluated as a function of the number of spacer units between the two nonnatural amino acids. The observed efficiency showed qualitative agreement with theoretical values calculated from interchromophore distances that were predicted from the helix geometry. When the biphenylnaphthyl pair is separated by a single amino acid, higher transfer efficiency than that predicted by theory was observed. The high efficiency was interpreted in terms of contributions from higher multipole interactions. An effect of a phenyl group inserted between the donor and acceptor was also studied. The intervening phenyl group slightly enhanced the energy transfer when the aromatic rings of the donor, mediator, and acceptor groups were nearly in a face-to-face configuration.