The recombinant nucleocapsid (rN) protein of the coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS) was cloned and expressed in Escherichia coli, extracted from cell lysates containing 6M urea, then purified by Ni(2+)-affinity chromatography. In animal immunogenicity studies, we found that most anti-rN protein antibodies were IgG2a in BALB/c mice vaccinated with rN emulsified in Montanide ISA-51 containing the synthetic oligodeoxynucleotide, CpG. In contrast, anti-rN protein antibodies of mice immunized with rN protein in PBS were found to mainly be IgG1. These results indicated that ISA-51/CpG-formulated rN protein was dramatically biased toward a Th1 immune response. To identify the B-cell immunodominant epitopes of the rN protein in the mouse and monkey, the reactivities of antisera raised against purified rN proteins formulated in ISA-51/CpG were tested with a panel of overlapping synthetic peptides covering the entire N protein sequence. Three immunodominant linear B-cell epitope regions were mapped to residues 166-180, 356-375, and 396-410 of the rN protein. When the reactivities of these peptides were screened with human sera from five SARS patients, peptides corresponding to residues 156-175 reacted strongly with sera from two of the SARS patients. These results indicated that the region around residues 156-175 of the N protein is immunogenic in the mouse, monkey, and human. We found that peptides corresponding to residues 1-30, 86-100, 306-320, and 351-365 contained murine immunodominant T-cell epitopes. To identify functional CTL epitopes of the N protein, BALB/c mice were immunized with peptides containing the H-2K(d) CTL motif emulsified in adjuvant ISA-51/CpG. Using an IFN-gamma secretion cell assay and analysis by flow cytometry, peptides containing residues 81-95 were found to be capable of stimulating both CD4(+) and CD8(+) cell proliferation in vitro. We also only observed that peptides corresponding to residues 336-350 were capable of stimulating IFN-gamma production in T-cell cultures derived from peripheral blood mononuclear cells (PBMCs) of macaques immunized with the rN protein emulsified in ISA/CpG adjuvant. Our current results together with those of others suggest that some immunodominant B-cell and T-cell epitopes are conserved in the mouse, monkey, and human. This information is very important for the development SARS diagnostic kits and a vaccine.