Puumala hantavirus (PUUV) is a rodent-borne agent causing nephropathia epidemica in humans, a milder form of haemorrhagic fever with renal syndrome occurring in Fennoscandia, central Europe and western Russia. In this study we characterized the immunogenicity of an E. coli-expressed nucleocapsid (N) protein of PUUV (strain Kazan-E6) in inbred mice (BALB/c, CBA and C57/BL6). The recombinant N (rN) protein raised PUUV-specific antibodies in all three tested murine haplotypes, and all IgG subclasses were detected. Epitope mapping using peptides spanning the N protein revealed that the B-cell recognition sites were mainly located at the amino-terminal part of the protein. Proliferative T-helper (Th) lymphocyte responses were detected in all haplotypes after a single immunization with rN. Several Th-recognition sites, spanning amino acids 6-27, 96-117, 211-232 and 256-277, were identified using overlapping peptides. Peptides representing the identified sites could also prime Th-lymphocytes to proliferate in response to recall with rN protein, thereby confirming the authenticity of the identified sites. The rN-primed Th-lymphocytes produced predominantly interleukin (IL)-2 and gamma interferon, together with lower levels of IL-4 and IL-6, indicating a mixed Th1/Th2 response.