Helicobacter pylori colonizes the mucous layer of the stomach and the surface of gastric mucous cells. Although H. pylori is not generally thought of as invasive, it has been observed in the lamina propria and within vacuoles in the cytoplasm of epithelial cells. The authors report that isolates of H. pylori can enter into the cytoplasm of tissue culture epithelial cell lines such as HEp-2 cells. Intracellular uptake of H. pylori by HEp-2 cells is rapid and appears to require both the N-acetylneuraminyllactose-binding adhesin and another factor present only in living bacteria. Uptake of H. pylori was inhibited by ammonium chloride and chloroquine at concentrations that did not effect either adherence or bacterial viability. Dansylcadaverine, an inhibitor of receptor clustering and internalization, also inhibited uptake but not adherence of H. pylori. Uptake was completely inhibited when H. pylori and HEp-2 cells were incubated at 4 degrees C under conditions that did not effect bacterial adherence. Cytochalasin B, an inhibitor of phagocytosis, did not inhibit uptake. It is concluded that H. pylori is internalized either by receptor-mediated endocytosis or by a closely related pathway.