The present study was carried out to evaluate the immunogenicity and protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae, by cloning the full-length DnaJ of S. pneumoniae and expressing in heterologous host E. coli BL-21 (DE3). PCR amplified DnaJ was ligated in pQE-30 expression vector and subsequently transformed in E. coli DH5alpha strain. Cloning of DnaJ was confirmed by double digestion and PCR, followed by DNA sequencing. The His-tag containing recombinant protein was purified by Ni-NTA affinity chromatography. To determine the immunogenicity of DnaJ, the mice (10 mice/group) were immunized by injecting 40 microg DnaJ protein/mouse i.p. There was a significant increase in IgG titres (2 x 10(5)) in mice immunized with DnaJ protein. Isotyping studies revealed that antibodies produced are predominantly IgG2a type indicating the predominance of Th1 response. A significant increase in lymphocyte proliferation was observed in mice immunized with DnaJ protein as compared to the control mice. Further, there was a significant increase in IL-2 and gamma-IFN levels in culture supernatants of splenocytes isolated from immunized mice. To determine the efficacy of DnaJ vaccination in eliciting protection, the mice were challenged with 1 x 10(5)cells of S. pneumoniae A66 type 3 capsular strain intra-nasally after 7 days of last immunization. All the control mice died within 2 days of post-infection, while 70% of animals immunized with DnaJ survived the lethal challenge by S. pneumoniae. The study reveals that immunization of mice with DnaJ elicits protective immunity against S. pneumoniae infection.