Cholera toxin and Escherichia coli heat-labile enterotoxin are powerful mucosal adjuvants but their high toxicity hampers their use in humans. Site-directed mutagenesis has allowed the generation of several cholera toxin and E. coli heat-labile enterotoxin mutants with abolished or strongly reduced toxicity that still retain strong mucosal adjuvanticity. Among them, LTK63 (Ser to Lys substitution at position 63 in the A subunit) is completely nontoxic and LTR72 (Ala to Arg at position 72) retains a very low residual enzymatic activity. Both of them have been shown to be safe and effective in enhancing the immunogenicity of intranasally coadministered vaccines, also resulting in protective responses in several animal models. Clinical grade preparations of these mutants have now been produced, tested in animals and proven to be totally safe. Indeed, they did not induce any inflammatory event in the respiratory tract nor, more importantly, in the olfactory bulbs and in the meninges. The fully nontoxic LTK63 mutant has now been successfully tested in human volunteers with a trivalent subunit influenza vaccine.