The adherence of S. pneumoniae to human type-II pneumocytes and endothelial cells (EC) is critical to the pathogenesis of pneumococcal pneumonia and bacteremia. We established that the preferred target cell to which pneumococci adhere in the lung is the type-II lung cell (LC) and have developed an in vitro adherence assay to determine the molecular details of this interaction. Pneumococcal receptors on cultured human LC and EC appeared to be glycoproteins since treatment of the monolayers with tunicamycin significantly impaired bacterial adherence. Inhibition of adherence to LC and EC occurred following incubation with several carbohydrates including GalNAc, mannose and GalNAc beta-4Gal- and GalNAc beta 1-3Gal-containing glycoconjugates. Pneumococci could bind directly to these immobilized sugars and their addition to adherent pneumococci could elute the bacteria from LC and EC. Combinations of glycoconjugates indicated that two independent classes of pneumococcal receptor existed on both cell types. These were defined by the minimal receptor units GalNAc beta 1-4Gal and GalNAc beta 1-3Gal which participate in pneumococcal cell wall and protein-dependent mechanisms of adherence, respectively.