Prestin belongs to the SLC26 protein family, which regroups anion antiporters capable of transporting monovalent and divalent anions across biological membranes. Also referred to as SLC26A5, prestin is a motor protein essential for the electromotility of the outer hair-cells (OHC) and therefore the amplification of sound in the cochlea. The diffusion of prestin in the membrane has been previously studied through fluorescent recovery after photobleaching (FRAP) experiments, in HEK293 cells. We were able to determine that up to 50% of the prestin population was immotile. This suggest that intermolecular interactions between prestin, the membrane and the cytoskeleton are essential for prestin organization and function. We have created transgenic mouse lines co-expressing prestin-TFP and prestin-YFP. OHCs isolated from these mice have prestin-induced non linear capacitance (NLC) and electromotility comparable to wild-type mice.FRAP analysis on prestin-YFP indicated that in OHCs, the entire prestin population is immotile. This motility was partially recovered by inhibition of the actin filament polymerization. Fluorescent resonance energy transfer (FRET) coupled to fluorescence lifetime imaging microscopy (FLIM) allowed us to detect and monitor the prestin-prestin interactions at the nanometer scale. These FLIM-FRET experiments revealed a FRET efficiency of 25-35%.The FRAP experiments suggest a strong interaction of prestin with other membrane proteins or the cytoskeleton, and the high FRET efficiency will allow for prestin-prestin interactions to be monitored during alterations of the membrane composition and potential.