Streptococcus pneumoniae (the pneumococcus) continues to account for significant morbidity and mortality worldwide, causing life-threatening diseases such as pneumonia, bacteremia and meningitis, as well as less serious infections such as sinusitis, conjunctivitis and otitis media. Current polysaccharide vaccines are strictly serotype-specific and also drive the emergence of non-vaccine serotype strains. In this study, we used microarray analysis to compare gene expression patterns of either serotype 4 or serotype 6A pneumococci in the nasopharynx and blood of mice, as a model to identify genes involved in invasion of blood in the context of occult bacteremia in humans. In this manner, we identified 26 genes that were significantly up-regulated in the nasopharynx and 36 genes that were significantly up-regulated in the blood that were common to both strains. Gene Ontology classification revealed that transporter and DNA binding (transcription factor) activities constitute the significantly different molecular functional categories for genes up-regulated in the nasopharynx and blood. Targeted mutagenesis of selected genes from both niches and subsequent virulence and pathogenesis studies identified the manganese-dependent superoxide dismutase (SodA) as most likely to be essential for colonization, and the cell wall-associated serine protease (PrtA) as important for invasion of blood. This work extends our previous analyses and suggests that both PrtA and SodA warrant examination in future studies aimed at prevention and/or control of pneumococcal disease.