Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool to study airway biology. In this study we have investigated whether this culture system produces `mucus` with a similar protein composition to in vivo, induced airways secretions. Previous compositional studies on mucous secretions have greatly under-represented the contribution of mucins which are major structural components of normal mucus. To overcome this limitation we have used a mass spectrometry based approach centered upon prior separation of the mucins from the majority of the other proteins. Using this approach we have compared the protein composition of apical secretions (AS) from well differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions with host defense proteins being predominant. The epithelial mucins MUC1, MUC4 and MUC16 and the gel forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed the gel forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells there was considerable similarity between the two secretions; in particular in the category of host defense proteins which includes the mucins. This shows that the primary cell culture system is an important model to study aspects of innate defense of the upper airways related specifically to mucus comprised solely of airway cell products.