The fragmentation of 23S rRNA of 23 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112bp long intervening sequence (IVS) at position 542 and a conserved 121-123bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.