The effectiveness and safety of known mutants of Escherichia coli heat-labile enterotoxin (LT) as an adjuvant for nasal influenza vaccine were examined. Six mutants, called LT7K (Arg to Lys), LT61F (Ser to Phe), LT112K (Glu to Lys), LT118E (Gly to Glu), LT146E (Arg to Glu) and LT192G (Arg to Gly) were constructed by the replacement of one amino acid at one position of the A1 subunit to another using site-directed mutagenesis. All mutants were confirmed to be less toxic than wild-type LT when analyzed using Y-1 adrenal cells in vitro. When influenza vaccine was administered intranasally with LT7K and LT192G, BALB/c mice developed high levels of serum and local antibodies to the HA molecules. The adjuvant activity of these mutant LTs corresponded to that of wild-type LT when 1 microgram of these mutant LTs (or wild-type LT) was coadministered with the vaccine. From the point of view of safety, LT7K was considered to be the most potent mucosal adjuvant and was examined in more detail. The adjuvant activity of the mutant was lowered more rapidly with a decrease in dose than was that of wild-type LT. The low level of adjuvant of a relatively small amount of LT7K was heightened by adding LTB to the mutant LT. These results suggest that LT7K supplemented with LTB can be used as a less toxic, effective adjuvant for nasal influenza vaccine.