Pyochelin (PCH) is a siderophore produced and secreted byPseudomonas aeruginosafor iron capture. Using 55Fe uptake and binding assays, we showed that PCH-Fe uptake inP. aeruginosainvolves, in addition to the highly studied outer membrane transporter FptA, the inner membrane permease FptX, which recognizes PCH-55Fe with an affinity of 0.6 ± 0.2 nM and transports the ferri-siderophore complex from the periplasm into the cytoplasm:fptXdeletion inhibited 55Fe accumulation in the bacterial cytoplasm. Chromosomal replacement was used to generateP. aeruginosastrains producing fluorescent fusions with FptX, PchR (an AraC regulator), PchA (the first enzyme involved in the PCH biosynthesis) and PchE (a non-ribosomic peptide-synthetase involved in a further step). Fluorescence imaging and cellular fractionation showed a uniform repartition of FptX in the inner membrane. PchA and PchE were found in the cytoplasm, associated to the inner membrane all over the bacteria and also concentrated at the bacterial poles. PchE clustering at the bacterial poles was dependent on PchA expression, but on the opposite PchA clustering and membrane association was PchE-independent. PchA and PchE cellular organization suggests the existence of a siderosome for PCH biosynthesis as previously proposed for pyoverdine biosynthesis (another siderophore produced byP. aeruginosa).