Abstract.-Electronic excitation energy can be transferred between chromophores separated by distances of the order of 30 A. Forster proposed that the transfer occurs by a dipole-dipole resonance interaction which depends on certain spectroscopic and geometric properties of the donor-acceptor pair. His prediction that the rate of transfer depends on the inverse sixth power of the distance between the chromophores was verified previously. In this work, we tested a second prediction of F6rster's theory, namely, that the transfer rate is proportional to J, the magnitude of the overlap between the emission spectrum of the energy donor and the absorption spectrum of the energy acceptor.The energy donor was an N-methylindole moiety, and the acceptor was a ketone. These chromophores were fused to a rigid steroid that separated them by 10.2 A. Rate constants for singlet-singlet energy transfer in this system were obtained by nanosecond flash spectroscopy. J was varied over a 40-fold range simply by altering the solvent. We found that the transfer rate is proportional to J, as predicted by F6rster's theory. The results bear on the potential use of this energy transfer process to measure distances in biological macromolecules. It is evident that the length of such a spectroscopic ruler can readily be controlled by varying the magnitude of the spectral overlap integral of the energy donor-acceptor pair.Singlet-singlet energy transfer can occur over distances of the order of 30 A. 13 Forster has proposed that the transfer occurs by a dipole-dipole resonance interaction between the energy donor and acceptor chromophores. In his theory, the rate constant for transfer kT is related to geometric and spectroscopic factors by5 kT = r-JK2 n-4 kF X 8.71 X 1023 sec-1where r is the distance (in A) between the centers of the donor and acceptor transition moments, K2 is the dipole-dipole orientation factor, n is the refractive index of the medium, and kF is the rate constant (in sec1) for fluorescence emission by the donor. The spectral overlap integral J, which measures the extent to which the donor and acceptor transitions are in resonance, is given by = fF(X)e(X)dX (2) where F (X) is the fluorescence intensity of the energy donor at wavelength X (in cm), and E(X) is the molar decadic extinction coefficient (in cm-' M-) of the energy acceptor.