LeuT mutants were expressed in E. coli, purified, and labeled on targeted engineered cysteines with Cy3 and Cy5 maleimide. The functional properties of the labeled constructs were determined by measuring Leu binding by scintillation proximity assay, and Ala transport was measured after reconstitution of the protein into proteoliposomes. The fluorescence properties of labeled proteins were studied to establish specific and efficient labeling and to establish that the observed FRET changes likely arise from inter-dye distance rather than photophysical phenomena. Various constructs were created, each with two cysteine residues strategically placed for labeling. Purified, labeled protein was immobilized onto a passivated-glass surface via a streptavidin-biotin linkage. Fluorescence data were acquired using a prism-based total internal reflection (TIR) microscope. Fluorescence resonance energy transfer (FRET) efficiency was calculated and analysis of fluorescence and FRET traces was achieved using automated analysis software developed for this application. The single molecule traces were analyzed for LeuT in the presence and absence of the substrates sodium and Leu, upon addition of the transport inhibitors clomipramine and octylglucoside, and in response to mutations of the extracellular vestibule as well as the network of intracellular residues proposed to stabilize the inward closed state. Molecular dynamics simulations of the protein immersed in an explicit membrane, solvated with water molecules, ions and ligands, were carried out and long equilibrations (totaling >500 ns) were run to assess conformational changes.