A cluster of 14 genes located on the large plasmid of enteropathogenic Escherichia coli (EPEC) strains is sufficient to direct the biogenesis of the type IV bundle-forming pilus (BFP) in a recombinant E. coli host. The fifth gene in the cluster, bfpU, encodes a protein that is predicted to be localized to the periplasmic space. To determine whether BfpU is necessary for pilus biogenesis, the authors constructed a non-polar bfpU mutant EPEC strain by allelic exchange. The mutant strain was unable to perform localized adherence and auto-aggregation, two phenotypes associated with BFP expression, and it failed to make BFP. These phenotypes were restored to the bfpU mutant by a plasmid containing bfpU. There was no difference between the wild-type and bfpU mutant strains in their expression or processing of the pre-pilin protein or in their localization of the pilin protein in the inner and outer membranes. Fractionation studies revealed that BfpU is completely soluble and is detected in both the periplasm and the cytoplasm. Thus, BfpU represents a novel protein required for type IV pilus assembly.