To determine the kinetics of ascension of the eustachian tube (ET) by non-typeable Haemophilus influenzae (NTHi) in situ from the nasopharynx to the middle ear using an experimental model of otitis media (OM), we examined snap-frozen sections of chinchilla ET and middle ear mucosa for adherent bacteria over a 14 day time period. Via fluorescent- and transmission electron-microscopy, we found that NTHi preferentially adhered not to the epithelial cells but to the mucus in the ET and gradually ascended this tubal organ, reaching the middle ear approximately 10 days after intranasal inoculation of adenovirus-infected animals. The number of NTHi adherent to mucus at the pharyngeal portion of the ET increased significantly in the first 4 days after inoculation of the nares whereas the number of adherent bacteria in both the mid and tympanic portions of the ET increased more gradually over time. NTHi were not observed in the middle ear until approximately 7-10 days after inoculation of the nares which was coincident with the onset of clinical signs of OM. These data confirmed our earlier in vitro investigation which suggested that adherence to and growth within stagnant mucus within a ET compromised by adenovirus was a possible mechanism by which NTHi, resident in the nasopharynx, might gain access to the middle ear and induce OM.