Haemophilus influenzae has the ability to obtain iron from human transferrin via two bacterial cell surface transferrin binding proteins, Tbp1 and Tbp2. Although a wide array of strains have been shown to express these receptor proteins, two studies have recently identified a series of isolates which appeared to lack the ability to bind transferrin. Included in this group were the members of a cryptic genospecies of nontypeable biotype IV strains which appear to possess a tropism for female urogenital tissues and are major etiologic agents of neonatal and postpartum bacteremia due to H. influenzae. The present study employed oligonucleotide primers specific for genes encoding the Tbp proteins of a type b biotype I strain of H. influenzae to probe the genomic DNAs of isolates from the previous studies. The tbpA and tbpB genes which encode Tbp1 and Tbp2, respectively, were detected in all of the strains tested either by PCR amplification directly or by Southern hybridization analysis. All of the strains displayed a transferrin binding phenotype, and affinity isolation of receptor proteins with transferrin-conjugated Sepharose recovered Tbp1 and/or Tbp2 from 11 of 14 strains, including 2 of the nontypeable biotype IV strains. In addition, all of the strains were capable of growing on human transferrin specifically, indicating that the mechanism of iron assimilation from transferrin is functional and is not siderophore mediated. These results confirm the presence of tbp genes in all of the invasive H. influenzae isolates characterized to date, suggesting that Tbp-mediated iron acquisition is important in disease which initiates from either the respiratory or urogenital mucosa.