Non-typeable Haemophilus influenzae (NTHi) and encapsulated Haemophilus influenzae type b (Hib) were inoculated into the middle ears of Sprague-Dawley rats. Tympanic membrane (TM) status was assessed otomicroscopically and specimens from various middle ear areas were prepared for light microscopy at various times during the acute phase and up to 6 months after inoculation. Irrespective of bacteria strain, acute otitis media (AOM) was present in all ears 4 days after inoculation. The Hib-infected ears showed initially a severe course of AOM, but all were otomicroscopically resolved by day 12, at which time a few NTHi-inoculated ears still exhibited middle ear effusion. The TMs infected with Hib had normalized without scar formation, whereas NTHi induced a persistent thickening of the TMs in half of all cases. The middle ear mucosa of NTHi-infected ears initially showed vigorous activity among the goblet cells, but the mucosa normalized after the acute phase. Hib, by contrast, induced prominent changes in the middle ear mucosa. Initially, no goblet cell granules or ciliated cells could be observed in the mucosa. Later on, the epithelium contained large, active goblet cells. Glands appeared beneath the mucosa which persisted as streaks of epithelial cells throughout the study period. The findings show that NTHi and Hib both induce AOM but with differing clinical courses, and affect different targets in the middle ear.