To acquire iron, Pseudomonas aeruginosa secretes a major fluorescent siderophore, pyoverdine (PvdI), that chelates iron and shuttles it into the cells via the specific outer membrane transporter, FpvAI. We took advantage of the fluorescence properties of PvdI and its metal chelates as well as the efficient FRET between donor tryptophans in FpvAI and PvdI to follow the fate of the siderophore during iron uptake. Our findings with PvdI-Ga and PvdI-Cr uptake indicate that iron reduction is required for the dissociation of PvdI-Fe, that a ligand exchange for iron occurs, and that this dissociation occurs in the periplasm. We also observed a delay between PvdI-Fe dissociation and the rebinding of PvdI to FpvAI, underlining the kinetic independence of metal release and siderophore recycling. Meanwhile, PvdI is not modified but recycled to the medium, still competent for iron chelation and transport. Finally, in vivo fluorescence microscopy revealed patches of PvdI, suggesting that uptake occurs via macromolecular assemblies on the cell surface.