A genetic locus implicated in the development of competence in Haemophilus influenzae Rd has been previously mapped to a 12.8-kb PstI region of the chromosome [Tomb et al., J. Bacteriol. 171 (1989) 3796-3802]. To define the boundaries of this locus and to identify the gene(s) involved in transformation, additional mini-Tn10kan mutagenesis was performed and the region containing all mutagenic insertions was sequenced. Three new transformation-deficient (Tfo-) mutants were found, bringing the number of distinct mutations mapped to this region up to eight. The transformation frequency of strains carrying the new insertions was 25- to 10(5)-fold less than wild type. The ends of the mini-Tn10kan element were used as starting points to sequence a 9.1-kb region. The position of the eight mutagenic insertions was determined and ten putative open reading frames (ORFs) were found. One of the mini-Tn10kan elements had inserted in an intergenic region while the rest had inserted in six of the ORFs. Based on the phenotypes of the mutant strains and the position of the insertions, we concluded that at least three of the genes should be involved in transformation. In addition, fourteen 9-11-bp uptake signal sequences (USS) were found, four of which were part of stem-loop structures and could function as attenuators of terminators of transcription.