We evaluated the potential of B cell "superstimulatory" influenza viruses to activate peritoneal B cells (PBC) from BALB/c mice containing both CD5+ and CD5- "sister" cells. Like conventional B cells, PBCs responded to influenza viruses in a hemagglutinin glycoprotein (HA) subtype-specific manner with proliferation and vigorous Ig synthesis. However, a number of HA subtypes that are highly stimulatory for conventional B cells failed to induce significant responses of PBC. Isotype-determination revealed a high predominance of IgM and only very low production of IgA and IgG. HA-activated CD5+ B cells showed a hyperexpression of various activation markers, including MHC class II, intercellular adhesion molecule 1 (CD54), and B7-1 molecules. In contrast to conventional B cells, where activation by HA is antagonized by phorbol esters (PMA), HA and PMA acted synergistically on PBC, suggesting differential activation requirements of B-2 cells vs PBC in response to HA. Like HA stimulation of B-2 cells, virus-triggered proliferation of PBC was abrogated by a simultaneous treatment with F(ab')2 fragments of anti-Ig Ab and exhibited synergistic effects with LPS stimulation. HA-mediated proliferative responses of PBC, but not of B-2 cells, were positively controlled by various cytokines, including IL-4 and IL-10, and to a lesser extent by IL-6. In conclusion, our data present the first example of a stimulation of peritoneal B cells by a polyclonal-activating virus, findings that call for considering infections with polyclonal B cell-stimulatory viruses as a means of expanding the pool of potentially autoreactive CD5+ B cells.