Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodelling of their components, called maturation. By using 2D gel electrophoresis and mass spectrometry, combined with genomic data, we have identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic and the cytoskeleton. By carrying out this proteomic analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Ga4 and Gß, appeared at the earliest times. We show that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.