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The biological functions of nucleic acids in processes of DNA replication, transcription, homologous recombination, mRNA translation, and ribozyme catalysis are intimately linked to their three‐dimensional structures and to conformational changes induced by proteins, metal ions and other ligands. Fluorescence spectroscopy is a powerful technique for probing the structure and conformational dynamics of biological macromolecules under a wide range of solution conditions. Fluorescence resonance energy transfer (FRET) provides long‐range distance information from 10 to 100 Å, a range that is useful for probing the global structure of nucleic acids. While steady‐state measurements of FRET provide the average distance between donor and acceptor, much more information is available from the analysis of the nanosecond emission decay of the donor in time‐resolved FRET (trFRET) experiments. Analysis of the decay in terms of donor–acceptor distance distributions can resolve different conformers in a heterogeneous mixture, providing information on the global structure and flexibility of each species as well as their equilibrium populations. In this review, we outline the principles of trFRET and the methods used to incorporate fluorescent probes into DNA and RNA. Examples of specific applications are presented to illustrate the versatility of trFRET as a tool to define global structures, to identify conformational heterogeneity and flexibility, to investigate the energetics of tertiary structure formation and to probe structural rearrangements of nucleic acids. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 159–179, 2002

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