Haemophilus influenzae can utilize iron-loaded human transferrin as an iron source for growth in vitro. H. influenzae tonB mutants, containing a chloramphenicol acetyltransferase gene within their tonB genes, could bind iron-charged human transferrin to their cell surfaces, but they were unable to utilize this serum glycoprotein as the sole source of iron for growth in vitro. In contrast, these tonB mutants were able to utilize an iron chelate (ferric ammonium citrate) for growth. Transformation of a tonB mutant with a plasmid encoding a wild-type H. influenzae tonB gene restored the ability of a tonB mutant to utilize iron-charged human transferrin. These results indicate that the uptake of iron from human transferrin by H. influenzae is a TonB-dependent process.