Single-molecule fluorescence spectroscopy is emerging as a powerful tool for detailed biophysical analyses of nucleic acid – protein interactions, because of the ability to resolve different binding modes and to provide kinetic information on protein conformational changes during assembly or biological function. To illustrate these capabilities, I will describe two systems currently under study in my laboratory. (1) The HIV-1 protein Rev mediates the nuclear export of unspliced and partially spliced mRNAs encoding viral structural proteins. Rev interacts with a highly conserved element within the viral pre-mRNA known as the Rev response element (RRE). This is a complex interaction in which multiple Rev monomers assemble on the RRE, mediated by a combination of RNA-protein and protein-protein interactions. Single-molecule TIRF microscopy is used to monitor hundreds of individual Rev-RRE assembly reactions in parallel on the surface of a quartz slide, revealing the mechanism of oligomeric assembly and the influence of cellular cofactors on the assembly pathway. (2) DNA polymerases replicate DNA substrates with extraordinarily high fidelity because of their ability to discriminate between cognate and non-cognate nucleotide substrates during each cycle of nucleotide incorporation and to remove misincorporated bases using a separate proofreading activity. Single-molecule FRET methods are used to observe conformational changes of the fingers subdomain of a model DNA polymerase during the process of selection of incoming nucleotide substrates, revealing a novel “ajar” conformation that acts as a fidelity checkpoint before the fingers enclose the nascent base pair. These methods are also used to monitor the movement of the nascent DNA strand during proofreading, revealing that the DNA can switch between the spatially separated polymerase and exonuclease sites while remaining bound to the enzyme. These observations provide new insights into the important role of enzyme conformational dynamics during the process of nucleotide substrate selection and proofreading.