The internalins InlA and InlC2 are encoded proteins from two strongly immunoreactive clones recently identified by differential immunoscreening of a Listeria monocytogenes serotype 4b genomic expression library during the search of the gene products of L. monocytogenes specifically induced in vivo during infection (Yu WL, Dan H, Lin M. J Med Microbiol 56:888-895, 2007). In this study, we examined the humoral immune response against InlA and InlC2 in various L. monocytogenes-infected hosts using Western blots. InlA and InlC2 were recognized by antibodies in experimentally infected rabbits but not by antisera from rabbits immunized with the heat-killed bacterium. Similar strong immunological reactions to InlA and InlC2 were seen with antisera from infected guinea pigs, cattle, and sheep but not with those from the animals (guinea pigs or sheep) receiving heat-killed bacteria. This study provides the first experimental evidence that InlA and InlC2 are the in vivo induced or upregulated antigens for humoral immune responses that are common to listerial infection of various host species. These two immunogenic proteins may thus be explored as reagents for the laboratory diagnosis of listeriosis or candidates for vaccine development.