Eukaryotic cells contain a large number of actin binding proteins of different functions, locations and concentrations. They bind either to monomeric actin (G-actin) or to actin filaments (F-actin) and thus regulate the dynamic rearrangement of the actin cytoskeleton. The Dictyostelium discoideum genome harbors representatives of all G-actin binding proteins including actobindin, twinfilin, and profilin. A phylogenetic analysis of all profilins suggests that two distinguishable groups emerged very early in evolution and comprise either vertebrate and viral profilins or profilins from all other organisms. The newly discovered profilin III isoform in D. discoideum shows all functions that are typical for a profilin. However, the concentration of the third isoform in wild type cells reaches only about 0.5% of total profilin. In a yeast-2-hybrid assay profilin III was found to bind specifically to the proline-rich region of the cytoskeleton-associated vasodilator-stimulated phosphoprotein (VASP). Immunolocalization studies showed similar to VASP the profilin III isoform in filopodia and an enrichment at their tips. Cells lacking the profilin III isoform show defects in cell motility during chemotaxis. The low abundance and the specific interaction with VASP argue against a significant actin sequestering function of the profilin III isoform.