The effectiveness of the two-dose regimen, composed of a primary intranasal inoculation of influenza A-type virus HA vaccine together with B subunit of cholera toxin (CTB) and the subsequent intranasal inoculation of vaccine alone 4 weeks later, was examined. In mice given a relatively high dose of virus A/PR/8/34 (PR-8, H1N1) HA vaccine (1.5 micrograms) both as a primary antigen with CTB (1 microgram) and as the second antigen, the secondary responses of both antiviral IgA antibodies in nasal wash and haemagglutination-inhibiting (HI) antibody in serum were much higher than those of primary responses and persisted for greater than 12 weeks after the second inoculation. Even in mice that received reduced amounts of a primary vaccine (0.03 microgram) [prepared from virus PR-8, A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2)] together with reduced amounts of CTB (0.05 microgram), the subsequent inoculation of PR-8 vaccine produced both nasal IgA and serum HI antibodies and provided complete protection against homologous A-type virus (PR-8) infection. Moreover, the combination of the reduced amounts of heterologous A-type virus vaccine (A/Yamagata or A/Fukuoka) with CTB for primary inoculation and the secondary heterologous A-type virus vaccine [A/Yamagata, A/Kumamoto/37/79 (H1N1), or A/Fukuoka] resulted in high levels of cross-reactive IgA antibodies and partial cross-protection against PR-8 infection. On the other hand, a second inoculation of B/Ibaraki/2/85 vaccine failed to produce cross-reactive antibodies and to protect against PR-8 infection.(ABSTRACT TRUNCATED AT 250 WORDS)