The type b capsule of pathogenic Haemophilus influenzae is a critical factor for H. influenzae survival in the blood and the establishment of invasive infections. Other pathogenic factors associated with type b strains may also play a role in invasion and sustained bacteremia, leading to the seeding of deep tissues. The gene encoding haemocin is the only noncapsular gene found to be specific to type b strains until now. Here we report the discovery of an approximately 16-kb genetic locus, HiGI1, that is present primarily in type b strains. Pulsed-field gel electrophoresis and Southern hybridization were used to map this new locus between secG (HI0445) and fruA (HI0446), which are contiguous in Rd, a nonpathogenic derivative of a serotype d strain. It is inserted at the 3' end of tRNA(4)(Leu) and has regions whose G+C content differs from the average genomic G+C content of H. influenzae. An integrase gene, which encodes a CP4-57 like integrase, is located downstream of tRNA(4)(Leu). Hybridization probes based on the sequences within the HiGI1 locus have been used to screen 61 H. influenzae strains (2 type a, 22 type b, 2 type c, 1 type d, 3 type e, 7 type f, and 21 nontypeable H. influenzae [NTHi]) from our collection. This HiGI1 locus exists in all 22 type b strains and two NTHi strains and is likely to have been acquired by an ancestral type b strain.