Legionella pneumophila, the agent of Legionnaires' disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.