Biogenesis of the type 1 pilus fiber in Escherichia coli requires the product of the fimC locus. We have demonstrated that FimC is a member of the periplasmic chaperone family. The deduced primary sequence of FimC shows a high degree of homology to PapD and fits well with the derived consensus sequence for periplasmic chaperones, predicting that it has an immunoglobulin-like topology. The chaperone activity of FimC was demonstrated by purifying a complex that FimC forms with the FimH adhesion. A fimC1 null allele could be complemented by the prototype member of the chaperone superfamily, PapD, resulting in the production of adhesive type 1 pili. The general mechanism of action of members of the chaperone superfamily was demonstrated by showing that the ability of PapD to assemble both P and type 1 pili was dependent on an invariant arginine residue (Arg-8), which forms part of a conserved subunit binding site in the cleft of PapD. We suggest that the conserved cleft is a subunit binding feature of all members of this protein family. These studies point out the general strategies used by Gram-negative bacteria to assemble adhesins into pilus fibers, allowing them to promote attachment to eukaryotic receptors.