Biotinyl-enzymes are conservative molecules present in helminths, as well as in other animals, bacteria and plants. They have recently been found to be antigenic in mice, and a potential source of cross-reactivity among helminths. This study investigated the presence in human sera of antibodies reactive with biotinyl-enzymes from the nematodes Anisakis simplex, Toxocara canis and Ascaris suum. Biotinyl-enzymes from all these nematodes were recognized by IgG1 antibodies in sera from healthy subjects and from Anisakis-free patients infected with other parasites. Interestingly, IgE antibodies reactive with Anisakis simplex biotinyl-enzymes were present in about one third of the sera from Anisakis-free patients infected with other parasites. Our results also demonstrate that the anti-BE IgG1 and IgE antibodies present in the sera of Anisakis-free subjects are cross-reactive among helminths. We conclude that biotinyl-enzymes from nematodes are recognized by natural human antibodies, although Anisakis biotinyl-enzymes do not seem to be the cause of sensitization. Since sera from the Anisakis-free population also present these antibodies, as-yet unidentified factors (dietary components, intestinal inflammation and/or the presence of parasites) may contribute to the induction of anti-BE antibody background.