Effects of the B subunit of cholera toxin (CTB) on the primary antibody responses to influenza virus A/PR/8/34 (PR-8) (H1N1) HA vaccine and on protection against viral challenge were investigated in Balb/c mice which were immunized intranasally with both the vaccine and CTB. The dose of CTB (greater than or equal to 1 microgram) inoculated with the vaccine (greater than or equal to 0.15 microgram) induced high responses of both antiviral IgA antibodies in the nasal wash and haemagglutinin-inhibiting (HI) antibody in the serum, enough to provide complete protection against viral challenge four weeks after immunization. High levels of antibody were maintained for more than 16 weeks after inoculation, affording complete protection during this interval. The inoculation of HA vaccine prepared from influenza viruses A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2) together with CTB provided partial protection against PR-8 infection, with production of antiviral IgA antibodies which were cross-reactive to PR-8 antigens whereas immunization with CTB and HA vaccine prepared from a different type of influenza virus (B/Ibaraki/2/85) failed to protect against PR-8 infection. These results indicate that CTB can produce an augmented and persistent antibody response to PR-8 HA vaccine, which is cross-protective to other A-type virus infections. The mechanisms by which CTB enhances the protective antibody responses to the nasally inoculated vaccine were investigated. The ability of CTB to augment antibody responses was lost, either when CTB was inoculated via the intravenous or subcutaneous route, or when CTB was introduced into nasal site one day before or after the vaccine inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)