Pneumococci traverse eukaryotic cells within vacuoles without intracytoplasmic multiplication. The platelet-activating factor receptor (PAFr) has been suggested as a portal of entry. Pneumococci colocalized with PAFr on endothelial cells and PAFr-/- mice showed a substantially impaired ability to support bacterial translocation, particularly from blood to brain. Pneumococci-induced colocalization of PAFr and β-arrestin 1 at the plasma membrane of endothelial cells and PAFr-mediated pneumococcal uptake in transfected COS cells were greatly increased by cotransfection with the scaffold/adapter protein β-arrestin 1. Activation of extracellular signal-regulated kinase kinases was required for uptake and was limited to the cytoplasmic compartment, consistent with activation by β-arrestin rather than PAFr. Uptake of the pneumococcal vacuole involved clathrin, and half the bacteria proceeded into vacuoles marked by Rab5 and later Rab7, the classical route to the lysosome. Overexpression of β-arrestin in endothelial cells decreased colocalization with Rab7. We conclude that the association of β-arrestin with the PAFr contributes to successful translocation of pneumococci. Copyright © 2005, American Society for Microbiology. All Rights Reserved.