Viral proteins are highly antigenic and known as potent stimulators of adaptive immune responses. This mechanism is often used for biotechnological applications in monoclonal antibody production resulting in high-affinity IgG antibodies in most cases. The aim of this study was to increase antigen-specific IgA antibody levels in mice in order to generate monoclonal IgA antibodies by hybridoma technology. For this purpose, hamster polyomavirus (HaPyV) major capsid protein VP1 was used to immunize mice by different routes in order to induce VP1-specific IgA titers. Recombinant HaPyV-VP1 was generated in Escherichia coli and administered intraperitoneally, orally, and intrarectally. VP1-specific antibodies were determined by ELISA in sera and organ culture supernatants. We found a significant increase of HaPyV-VP1-specific IgAs in spleen organ cultures after rectal immunization of mice but not in cultures of mesenteric lymph nodes, colon, or Peyer's patches. In contrast, oral and intraperitoneal immunization did not provide an appropriate specific IgA induction at all. These results show that specific IgA antibodies can be induced by intrarectal immunization in the spleen. The generation of monoclonal IgA antibodies with well-defined properties is a useful tool for the investigation of mucosal immune responses or autoimmune diseases and extends the spectrum of antibodies with specific effector functions.