Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several proteins and polysaccharide capsule have recently been implicated in the virulence of and protective immunity against Streptococcus pneumonia. Pneumococcal surface protein A (PspA) is highly conserved among S. pneumonia strains, inhibits complement activation, binds lactoferrin, elicits protective systemic immunity against pneumococcal infection, and is necessary for full pneumococcal virulence. Identification of PspA peptides that optimally bind human leukocyte antigen (HLA) would greatly contribute to global vaccine efforts, but this is hindered by the multitude of HLA polymorphisms. Here, we have used an experimental data set of 54 PspA peptides and in silico methods to predict peptide binding to HLA and murine major histocompatibility complex (MHC) class II. We also characterized spleen- and cervical lymph node (CLN)-derived helper T lymphocyte (HTL) cytokine responses to these peptides after S. pneumonia strain EF3030-challenge in mice. Individual, yet overlapping peptides, 15 amino acids in length revealed residues 199 to 246 of PspA (PspA(199-246)) consistently caused the greatest IFN-gamma, IL-2, IL-5 and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo stimulated splenic and CLN CD4(+) T cells isolated from S. pneumonia strain EF3030-challeged F(1) (B6xBALB/c) mice. IEDB, RANKPEP, SVMHC, MHCPred, and SYFPEITHI in silico analysis tools revealed peptides in PspA(199-246) also interact with a broad range of HLA-DR, -DQ, and -DP allelles. These data suggest that predicted MHC class II-peptide binding affinities do not always correlate with T helper (Th) cytokine or proliferative responses to PspA peptides, but when used together with in vivo validation can be a useful tool to choose candidate pneumococcal HTL epitopes.