An affinity procedure with purified, biotinylated human transferrin and streptavidin-agarose was used to identify the transferrin-binding proteins in strains of Haemophilus influenzae. Proteins of 58 and 98 Kda were isolated from total membranes prepared from iron-deficient but not iron-sufficient H. influenzae KC548 cells. The 58-Kda protein was capable of binding human transferrin after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electroblotting. Isolation of transferrin-binding proteins from type-b and non-typable H. influenzae strains demonstrated some variability in the size of the higher mol. wt protein (94-106 Kda) and in ease of elution of the smaller protein from the affinity resin. Use of purified, biotinylated human lactoferrin in the affinity isolation procedure with membranes from a strain expressing lactoferrin-binding activity resulted in isolation of proteins of 105 and 106 Kda distinct from the transferrin-binding proteins.