The impact of the 7-valent pneumococcal conjugate vaccine (PCV7) on the pneumococcal flora has been mostly studied without evaluating multiple colonization and the mechanism(s) leading to serotype replacement. These issues are addressed here, while assessing the effect of a single PCV7 dose A group of children received one PCV7 dose just after nasopharyngeal sampling, with the control receiving no vaccine and both groups being sampled again a month later Up to 10 pneumococcal isolates were recovered per colonized child-1224 isolates were serotyped and representative ones characterized by pulsed-field gel electrophoresis In vaccinated children. serotype replacement between vaccine (VT) and non-vaccine (NVT) types occurred in single and multiple carriers, and VTs were less prone to be de novo acquired. NVT unmasking was only detected in the vaccinated group One month after vaccination with a single dose. PCV7 prevents VT de novo acquisition and promotes NVT unmasking.