Pneumococcal surface protein A (PspA) is an immunogenic surface protein of Streptococcus pneumoniae. PspA of S. pneumoniae strain Rx1 is a 65-kDa protein composed of an α-helical N-terminus of 288 amino acids followed by an 82-amino-acid proline-rich region, 10 repeats of 20 amino acids each, and a 17-amino-acid C-terminus. It has been demonstrated that the 3'-half of pspA is relatively conserved among unrelated pneumococcal isolates and the 5'-half of the gene is highly variable. Additionally, nearly all pneumococcal strains contain at least one other locus with sequence homology to pspA. In this study oligonucleotides derived from the DNA sequence of pspA of Rx1 were used both as hybridization probes and as primers in the polymerase chain reaction (PCR) to investigate genetic variation within domains of pspA and in the pspA-like sequences from 18 strains representing 12 capsule and 9 PspA serotypes. Sequences encoding the leader peptide, the proline-rich region, and the repeat region are highly conserved among pspA and pspA-like sequences. The α-helical coding domain is highly diverse among pspA and pspA-like sequences of different strains.