A mutagenesis system involving the insertion of a non-transposable antibiotic resistance gene cassette was used to generate stable mutations in the chromosome of Haemophilus influenzae type b strain Eagan. The mutations generated were shown by pulsed-field gel electrophoresis (PFGE) to have unique SmaI fingerprint patterns and to be located randomly on the chromosome. Of 700 insertion mutants screened, 29 had stable insertions resulting in constitutive expression of transferrin-binding proteins (TBPs). The high proportion of such mutants indicated that numerous regulatory loci could influence the expression of this phenotype. Five such regulatory mutations were analysed in detail by PFGE and DNA hybridisation and were shown to be located at five different chromosomal loci, although three of the five loci were located on the same 330-kb SmaI fragment of the wild-type strain Eagan chromosome. This fragment also contains several important virulence determinants, including the capb locus, and one of the five constitutive mutants had concomitantly lost the ability to synthesise a type-b capsule. No DNA homology was demonstrated between H. influenzae chromosomal fragments separated by PFGE and DNA probes for the TBPs from Neisseria meningitidis, but the possibility of shared regulatory mechanisms controlling the expression of TBPs in these two species remains to be investigated.