The efficacy of pneumococal conjugate vaccines in young children may be complicated by serotype replacement. We developed a colony blot assay which enables the identification of re-colonization with novel serotypes (replacement), overgrowth by minor co-colonizing serotypes or suppression of previously predominant vaccine serotype strains as a result of vaccination. This method allows the identification of multiple serotypes in a single specimen in a ratio of 1:1000. In order to demonstrate the potential of our method, we investigated the consecutive nasopharyngeal samples of 26 children who had shown a shift in pneumococcal colonization after conjugate vaccination. Mixed colonization was found once in 15 pre-vaccination samples and four times in 26 post-vaccination samples. In the remaining children 'true replacement' had presumably occurred. Hence, we conclude that the colony blot assay is an easy to apply method, which allows the identification of different pneumococcal serotypes within single clinical specimens.