A putative pullulanase-encoding gene from Streptococcus pneumoniae was identified by screening a genomic expression library with human convalescent-phase serum. The 3,864-bp gene encoded a 143-kDa protein. Surface location and pullulanase activity of the protein, designated SpuA, was demonstrated. SpuA was present in all investigated pneumococcal isolates of different serotypes. The spuA 5' end was highly conserved among clinical isolates except for a 75-bp region. The properties of SpuA reported here indicate that this novel immunogenic surface protein might have potential as a vaccine target.