The entry of Listeria monocytogenes into the enterocyte-like Caco-2 cell line was studied as a function of cell polarization and differentiation. L. monocytogenes entered through the entire surface of nonpolarized cells and, predominantly, through the basolateral surface of polarized cells based on the following observations: (i) sites of L. monocytogenes invasion paralleled the distribution of the transferrin receptor, a well-known basolateral marker of polarization; (ii) numbers of internalized bacteria decreased dramatically when Caco-2 monolayers cultured beyond confluency were used (about 0.1% of the inoculated bacteria versus 1 to 2% with nonconfluent monolayers); and (iii) L. monocytogenes entry into postconfluent monolayers was greatly enhanced by treating cells with Ca(2)+ -free medium, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Ethylene glycol-bis (beta-aminoethyl ether)-N, N, N',N' -tetraacetic acid (EGTA) had contradictory effects on L. monocytogenes entry as this reagent opened intercellular junctions but inhibited binding and internalization of bacteria. Finally, the role of the inlAB locus in L. monocytogenes entry was confirmed because and inlAB mutant was 50- to 100-fold less invasive than the parental strain regardless of the monolayer's age. However, the inlAB mutant was still able to enter cells and to induce intracellular actin polymerization. Entry of inlAB bacteria into Caco-2 cells was not inhibited by EGTA.