Summary The pneumococcal cell surface protein PavA is a virulence factor associated with adherence and invasion in vitro. In this study we show in vivo that PavA is necessary for Streptococcus pneumoniae D39 colonization of the murine upper respiratory tract in a long-term carriage model, with PavA-deficient pneumococci being quickly cleared from nasopharyngeal tissue. In a pneumonia model, pavA mutants were not cleared from the lungs of infected mice and persisted to cause chronic infection, whereas wild-type pneumococci caused systemic infection. Hence, under the experimental conditions, PavA-deficient pneumococci appeared to be unable to seed from lung tissue into blood, although they survived in blood when administered intravenously. In a meningitis model of infection, levels of PavA-deficient pneumococci in blood and brain following intercisternal injection were significantly lower than wild type. Taken collectively these results suggest that PavA is involved in successful colonization of mucosal surfaces and in translocation of pneumococci across host barriers. Pneumococcal sepsis is a major cause of mortality worldwide so identification of factors such as PavA that are necessary for carriage and for translocation from tissue to blood is of clinical and therapeutic importance.