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We describe the theoretical basis of an unconventional method for the determination of the amount of energy transferred between two fluorophores by the F6rster mechanism. The method involves an internal comparison made by separation of the fluorophores in situ (i.e., in the optical cell), for example, by means of enzymic digestion; it eliminates several important sources of error and it simplifies calculation while making maximal use of the information contained in the fluorescence spectra. The validity of the method is demonstrated by determination of the known distance between two modifiable sites on the transfer RNA molecule, and its usefulness is exemplified by its application to triangulation of the ribosome of Escherichia coli.When a macromolecule is labeled with donor and acceptor fluorophores suitable for Forster-type energy transfer and the donor is excited, then the transfer of energy from donor to acceptor results in a quenching of the donor's emission and an enhancement of the acceptor's emission.The donor quenching is expressed experimentally by the ratio G of intensities in the presence and absence of the acceptor. Because the acceptor will normally have a finite extinction coefficient at the wavelength where the donor is excited, even if this is far from the excitation maximum of the acceptor, the acceptor enhancement can likewise be meaningfully expressed as the ratio H of intensities of acceptor fluorescence in the presence and absence of donor.The fraction of the excitation energy of the donor transferred to the acceptor is defined as E, and this is related to the observables G and H by the standard equations According to Forster (1), the distance R between the fluorophores is then given by R = Ro -1) [3] in which Ro is the distance between the fluorophores when E = 0.5. The problems associated with measuring Ro are well known (2-4); however, considerable problems are associated also with the determination of G and H, and it is with these that this paper is concerned. The measurement of G and H requires, as stated above, the determination of the ratios of intensities of donor (or acceptor) emission with and without acceptor (or donor). These are in principle easily obtained by comparing the spectra of doubly labeled (donor and acceptor) and of singly labeled (donor or acceptor) macromolecules; as a rule, the spectrum of the unlabeled macromolecule is used as a background correction. In practice, however, the comparison of these intensities is accompanied by three sources of potentially serious experimental error. (i) The concentrations of the four samples (doubly labeled, singly labeled, and unlabeled) must be either exactly equal or else accurately known, for normalization. (ii) The degrees of labeling of both singly and doubly labeled samples must be known and should preferably be identical. (iii) The observation of the intensity ratios in most cases is complicated by the fact that the emission spectra of donor and acceptor overlap at least partially and sometimes (e.g., for the freq...

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