Pneumococcal surface protein A (PspA) is a virulence factor of Streptococcus pneumoniae that can elicit a protective antibody response. The pspA gene of strain Rx1 encodes a 65 kDa molecule composed of 588 amino acids. The N-terminal 288 amino acids are highly charged, and predict an a-helical coiled-coil protein structure. All monoclonal antibodies (MAbs) to PspA, obtained by screening against whole pneumococci, bind to the cc-helical region of PspA, suggesting that this region is surface exposed. The C-terminal 217 amino acids of PspA contain the surface anchor of PspA and does not appear to be cc-helical. In the middle of the molecule is a proline-rich region that is thought to traverse the cell wall. in this study we have mapped the immunogenic epitopes detected by 9 MAbs that were made against strain Rx1 PspA. Five of the MAb also react with the PspA of mouse virulent strain WU2. All epitopes were found in one of two portions of the a-helical region. One comprised the first 115 amino acids, and the other was within amino acids 192 and 260. The five MAbs that recognize WU2 PspA, but not the remaining four MAbs, were protective against strain WU2. The epitopes detected by four of the five protective MAbs mapped to region 192 to 260 of Rx1 PspA. The existence of protective epitopes in this region was confirmed by demonstrating that mice immunized with the cloned fragment containing these residues were protected from fatal infection with WU2. Since amino acids 192 to 260 are in the region of PspA anticipated to be adjacent to the cell wall, and probably well covered by capsule, the means by which antibodies to the region lead to protection is not obvious. © 1994 Academic Press, Inc.