Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrhea among infants in the developing world. The initial stage of EPEC pathogenesis involves the adherence of bacteria to epithelial cells in tight clusters, a pattern which has been termed localized adherence. EPEC carry 50-70 MDa plasmids, termed EPEC adherence factor (EAF) plasmids, which are sufficient to confer the localized adherence phenotype upon non-adherent E. coli strains. The EAF plasmid-encoded adherence factor has been demonstrated to be a type IV pilus which aggregates to form bundles and is thus termed the bundle-forming pilus (BFP). The gene encoding the major structural subunit of the BFP, bfpA, has been cloned from the EAF plasmid of EPEC strain E2348/69. In this dissertation, I describe the identification of the bfp gene cluster, a set of 14 essentially contiguous genes on the EAF plasmid, including bfpA and 13 downstream genes. While 10 of the proteins encoded by the genes of this cluster share sequence similarity with proteins involved in the biogenesis of other type IV pili, 4 are unique to this system. Expression of the 14 genes of the bfp gene cluster from an inducible, artificial promoter is sufficient for reconstitution of BFP biogenesis in a laboratory E. coli strain. Non-polar mutagenesis of 3 genes in the cluster, bfpU, bfpH, and bfpL, is described. Mutations in the bfpU and bfpL genes abolish BFP biogenesis, while a mutation in the bfpH gene has no detectable effect on BFP biogenesis or function. Using a monoclonal antibody raised against a BfpU-histidine fusion protein, BfpU is shown to be a periplasmic protein. This is the first evidence that a periplasmic phase of transport is involved in type IV pilus biogenesis. This well-defined system is currently being used to characterize the molecular mechanisms of type IV plus biogenesis in EPEC and should prove to be a useful model for advancing our understanding of type IV pilus biogenesis in many other important human pathogens as well.