Nontypeable Haemophilus influenzae (NTHi) is sometimes the causative agent of invasive diseases, and it has been suggested that there may be differences in virulence among NTHi strains. Whilst studying clinical isolates of NTHi in a rat model of acute otitis media, intra- and interstrain differences in virulence were observed. Two strains with suddenly reduced capacity to cause middle ear infections and one highly virulent strain with dose requirements comparable only to encapsulated H. influenzae strains were further investigated, together with 15 other H. influenzae strains. The strains were characterized by analyzing the lipopolysaccharide, the outer membrane proteins, the hemagglutinating ability, and the polymerase chain reaction products after amplification of a gene sequence associated with encapsulation. The pathogenic capacity was assessed in two different in vivo models. It was found that the two strains with reduced pathogenic capacity could regain their virulence after animal passage. The LPS analysis and the results from the chicken embryo model suggested that the observed change in virulence might be associated with the lipopolysaccharide. For the non-animal-passaged strain 3655 there were indications that an undefined factor(s) contributed to its relatively potent virulence. As all three strains lacked genes necessary for encapsulation, in no case could any part of the increased virulence be attributed to the expression of small amounts of capsule.