Klebsiella pneumoniae obtained from patients with urinary tract infections is able to invade cultured human epithelial cells. The internalization process is dependent upon both microfilaments and microtubules. To better understand the interaction of these invasive bacteria with the host cell receptor(s), bladder, lung, and ileocecal epithelial cells were infected with K. pneumoniae in the presence of various lectins possessing multiple glycan specificities. It was found that the N-acetylglucosamine (GlcNAc)-specific lectins concanavalin A, Datura stramonium agglutinin, and wheat germ agglutinin significantly inhibited the invasion of K. pneumoniae into these cells but did not interfere with the internalization of an invasive strain of Salmonella typhimurium. Conversely, internalization of K. pneumoniae but not S. typhimurium was also significantly inhibited when the bacteria were pretreated with GlcNAc or chitin hydrolysate, a GlcNAc polymer, prior to the gentamicin invasion assay. Other carbohydrates such as glucose, galactose, mannose, fucose, and N-acetylneuraminic acid had no inhibitory effects on K. pneumoniae uptake. Furthermore, internalization of K. pneumoniae but not S. typhimurium by HCT8 cells was also significantly inhibited when eukaryotic protein glycosylation was interrupted by tunicamycin or when host N-linked surface glycans were removed by pretreatment with N-glycosidase F. These studies suggest that a N-glycosylated protein receptor is involved in the internalization of K. pneumoniae by human epithelial cells in vitro. The results also indicate that internal GlcNAc residues might be a carbohydrate component of the receptor.