Aggregation substance (AS), a surface protein encoded on the pheromone-inducible plasmids of Enterococcus faecalis, has been shown to increase adherence and internalization into a number of different cell types, presumably through integrin binding mediated by the N-terminal RGD motif of AS. Here, defined mutations constructed in Asc10, the AS encoded by the plasmid pCF10, are analyzed for their ability to promote increased internalization levels into HT-29 enterocytes. The results clearly show that the previously identified Asc10 functional domain, not the RGD motifs, is critical for Asc10-directed internalization of E. faecalis into HT-29 enterocytes. Also, expression of Asc10 in the nonaggregating E. faecalis strain INY3000 is unable to mediate HT-29 internalization. However, Asc10-expressing E. faecalis cells are not internalized as bacterial aggregates, suggesting bacterial aggregation is not a prerequisite for HT-29 internalization. These data show that Asc10 directs internalization of E. faecalis into HT-29 enterocytes through a non-RGD-dependent mechanism.