The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5' termini of a series of doublestranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of doublestranded B-DNA structure; the helical geometry of doublestranded DNA in solution is clearly evident. The experimental data were consistent with a 1/[1 + (RIRo)6] dependence of FRET efficiency characteristic for the Forster dipole-dipole mechanism. The thermal dissociation of the strands of the duplex DNA species can be followed by using FRET, and from these data we have been able to obtain enthalpies of duplex formation in good agreement with earlier measurements using alternative techniques. FRET measurements at very different salt concentrations can be accurately compared. We conclude that FRET is a reliable and valuable method for studying structure and conformational transitions in nucleic acids.Nucleic acids may adopt specific and sometimes complex folded structures that are critical for their biological function. Full determination of these structures requires the measurement of distances up to 80 A or more, but there are few techniques that allow such distances to be determined in solution. This is particularly important for nucleic acids in view of the extended helical structures involved, which can lead to underdetermination of structures by methods that can only yield short distances. Fluorescence resonance energy transfer (FRET) is sensitive to distances in the longer size range and has recently proved to be very useful in the study of nucleic acid structures, such as the solution structure of the four-way DNA junction (1, 2). FRET-derived distance information in DNA and RNA could be complementary to the shorter distances determined by NMR. However, the application of FRET to fluorescence probes covalently linked to nucleic acid structures has been relatively infrequent (1,(3)(4)(5)(6)(7)(8)(9)(10)(11).The rate of nonradiative energy transfer from an excited donor molecule (D) to a nearby acceptor molecule (A) depends in a characteristic manner on the distance between the two chromophores and their relative angular disposition (12)(13)(14). Depending on the D-A molecular pairs, the efficiency of transfer responds sensitively to relatively small changes of distances in the range of 10 to 80 A. Careful evaluations of FRET experiments can yield quantitative estimates of distances between labeled positions in macromolecules, or in molecular aggregates, provided that certain spectroscopic parameters are known. Even if sufficient information is not available to calculate exact distances, relative dimensions of molecular structures can often be deduced (1, 2, 11), and in principle FRET is applicable to very complex molecular structures. The method has been applied successfully to estimate intra(inter)molecular distances between donor and acceptor molecules in biological mo...