A diffusion-enhanced energy transfer technique was employed for the determination of transmembrane location of the retinal chromophore in the purple membrane. Theoretical considerations showed that the rate of energy transfer from an energy donor embedded within a membrane to acceptors dissolved in solvent could be described by an analytical function of the distance a of closest approach between the donor and acceptor, if the "rapid-diffusion limit" was attained. The criterion for this limit was given by the relation: (RO)6 much less than 20D tau Da4, where RO is the characteristic distance of energy transfer, D is the diffusion coefficient of the acceptor and tau D is the fluorescence lifetime of the donor in the absence of acceptor. By photo-reduction of the purple membrane with sodium borohydride, the retinal chromophore was converted to a highly fluorescent derivative, which showed a broad emission band in the visible region. From analysis of the fluorescence decay curves of the photo-reduced purple membrane in the presence of various concentrations of cobalt-ethylenediamine tetraacetate (Co-EDTA: energy acceptor), the depth of the chromophore from the membrane surface was estimated to be 8 (+/-3) A. This result was supported by investigations of energy transfer processes in a system where the native purple membranes and the photo-reduced membranes were stacked in parallel: the energy acceptor in this system was the native retinal chromophore.