Nontypable Haemophilus influenzae and Moraxella catarrhalis are important pathogens in children and adults. The mechanisms of their adherence to the epithelial cell surface and colonization are not clear. For the pathogen to adhere to the epithelial cell, it must first attach to and penetrate the mucus barrier. Mucin glycoproteins of the mucus layer generally are thought to be involved in bacterial attachment. To understand the precise mechanisms of middle ear mucin-bacterial interactions, we used an overlay binding assay with a highly purified middle ear mucin and outer membrane proteins of both nontypable H. influenzae and M. catarrhalis. Outer membrane proteins P2 and P5 were identified as the major components that medicate the binding between nontypable H. influenzae and human middle ear mucin. Moreover, the 57 kDa protein, CD, of the outer membrane protein of M. catarrhalis was found to be the only protein binding human middle ear mucin. Finally, it appears that a protein-oligosaccharide interaction is responsible for binding because asialo-mucin does not bind to either of the bacteria. Knowledge of the specific bacterial-mucin interaction may provide an understanding of the bacterial-epithelial cell colonization. Conversely, comprehension of this interaction between bacteria and purified mucin may be a strategy to prevent colonization of potential pathogens that cause otitis media and sinusitis in children.