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Prokaryotic aspartate transporters belong to the large family of cation-coupled membrane transporters. Members of this family are involved in several essential processes such as clearance of neurotransmitters from the synaptic cleft, or the uptake of amino acids in prokaryotes. These transporters form obligate trimers that arrange into a bowl-shaped assembly. Several crystal structures are available (Boudker et. al. 2007, Yernool et. al. 2004) which have revealed that large conformational changes take place during substrate translocation. Notably, a recent structure has captured the transporter in an asymmetric configuration with one subunit having a very different conformation from the others (Verdon and Boudker, 2012). However, these structures only provide a static picture of the transport cycle. To fully understand the transport cycle and subunit dynamics of these transporters, it will be necessary to monitor the conformational changes in real time and at the level of individual subunits. Single-molecule techniques seem therefore ideal to answer the open questions regarding the glutamate transport family. We have probed the transport of aspartate through individual trimers of the archeal Na+ coupled aspartate transporter GltPh with single-molecule fluorescence and we were able to monitor the conformational changes associated with substrate translocation. These data allow us to refine our view of the different conformations involved in transport.