Streptococcus pneumoniae has proteins that are attached to its surface by binding to phosphorylcholine of teichoic and lipoteichoic acids. These proteins are known as choline-binding proteins (CBPs). CBPs are an interesting alternative for the development of a cost-effective vaccine, and PspA (pneumococcal surface protein A) is believed to be the most important protective component among the different CBPs. We sought to use CBPs eluted from pneumococci as an experimental vaccine. Since PspA shows variability between isolates, we constructed strains producing different PspAs. We used the nonencapsulated Rx1 strain, which produces PspA from clade 2 (PspA2), to generate a pspA-knockout strain (Rx1 ΔpspA) and strains expressing PspA from clade 1 (Rx1 pspA1) and clade 4 (Rx1 pspA4). We grew Rx1, Rx1 ΔpspA, Rx1 pspA1, and Rx1 pspA4 in Todd-Hewitt medium containing 0.5% yeast extract and washed cells in 2% choline chloride (CC). SDS-PAGE analysis of the proteins recovered by a CC wash showed few bands, and the CBPs PspA and PspC (pneumococcal surface protein C) were identified by mass spectrometry analysis. Subcutaneous immunization of mice with these full-length native proteins without adjuvant led to significantly higher rates of survival than immunization with diluent after an intranasal lethal challenge with two pneumococcal strains and also after a colonization challenge with one strain. Importantly, immunization with recombinant PspA4 (rPspA4) without adjuvant did not elicit significant protection.