Streptococcus pneumoniae is thought to adhere to the blood-brain barrier (BBB) endothelium prior to causing meningitis. The platelet activating factor receptor (PAFR) has been implicated in this adhesion but there is a paucity of data demonstrating direct binding of the bacteria to PAFR. Additionally, studies that inhibit PAFR strongly suggest that alternative receptors for pneumococci are present on the endothelium. Therefore, we studied the roles of PAFR and pIgR, an established epithelial pneumococcal receptor, in pneumococcal adhesion to brain endothelial cells in vivo. Mice were intravenously infected with pneumococci and sacrificed at various time points before meningitis onset. Co-localization of bacteria with PAFR and pIgR was investigated using immunofluorescent analysis of the brain tissue. In vitro blocking with antibodies and incubation of pneumococci with endothelial cell lysates were used to further probe bacteria-receptor interaction. In vivo as well as in vitro pneumococci did not co-localize with PAFR. On the other hand the majority of S. pneumoniae co-localized with endothelial pIgR and pIgR blocking reduced pneumococcal adhesion to endothelial cells. Pneumococci physically interacted with pIgR in endothelial cell lysates. In conclusion, bacteria did not associate with PAFR, indicating an indirect role of PAFR in pneumococcal adhesion to endothelial cells. In contrast, pIgR on the BBB endothelium may represent a novel pneumococcal adhesion receptor.