A protein with a relative subunit molecular weight of 81000 (81K) has been isolated in virtually pure form from the outer membrane of low iron grown cells of Escherichia coli K12. The 81K protein, which is part of the receptor complex for translocation of the siderophore ferric enterobactin, displays activity in vitro for binding both ferric enterobactin and colicin B. The dissociation constant for the 81K-ferric enterobactin compound at 4 degrees C in 2% Triton-0.1 M Tris, pH 7, was determined to be 10 nM. The N-terminal amino acid was identified as phenylalanine, and the amino acid composition was shown to be similar to that published for the ferric aerobactin-cloacin receptor of Enterobacter cloacae. A plasmid-bearing strain of E. coli was employed to confirm that degradation of 81K to a slightly smaller, inactive form (81K) is performed by a second outer membrane component, protein a. The endoproteolytic action of protein a was verified by the finding of alanine as the N-terminal residue of 81K. A survey of enteric species suggests that the 81K-protein a interaction is confined to the K12 strain of E. coli.