The spleen, bone marrow and lymph nodes are all known to be important organs for the initiation and maintenance of an immune response after vaccination. To investigate the differences and similarities in the humoral and cellular immune responses between these tissues, we vaccinated mice once or twice with the conventional human dose (15 microg HA) of influenza A (H3N2) split virus vaccine and analysed the sera and lymphocytes collected from the different sites. We found that the response of antibody secreting cells (ASC) in the lymph nodes appeared to be more transient than in the spleen, possibly because the influenza-specific IgM ASC in particular might have migrated from the lymph nodes immediately after activation. The serum antibody response was found to initially correspond with the ASC response elicited in the spleen and the lymph nodes, whereas the later serum IgG reflected the ASC response in the bone marrow. Proliferation of influenza-specific CD4(+) and CD8(+) cells was predominantly observed in the spleen and was associated with higher concentrations of cytokines than in the lymph nodes. The finding of influenza-specific CD8(+) cell proliferation in the spleen indicates that a split influenza virus vaccine may stimulate a cytotoxic T-cell response. Our results also showed that the primary response elicited a mixed Th1/Th2 profile, whereas the secondary response was skewed towards a Th2 type. Each of the three tissues had a different immunological pattern, suggesting that in preclinical vaccine studies, there is a case for investigating a range of immunological sites.