Enteropathogenic Escherichia coli (EPEC) adhere to tissue culture cells in a distinct pattern known as localized adherence (LA). We have defined two loci necessary for LA. A plasmid-encoded gene cluster encodes bundlin, the major structural subunit of a type-IV fimbria called the bundle-forming pilus (BFP), a prepilin peptidase necessary for processing of pre-bundlin to its mature form, and twelve other proteins. Under the control of an exogenous promoter, these 14 genes are sufficient for the biogenesis of BFP in a heterologous E. coli host. The chromosomal gene dsbA, which encodes a periplasmic disulfide-bond oxidoreductase, is also required for LA. In the absence of DsbA protein, bundlin is made but rapidly degraded. Pre-bundlin is also rapidly degraded in the absence of DsbA, suggesting that the prepilin is a transcytoplasmic protein simultaneously accessible to enzymes on both sides of the inner membrane. These studies offer a fresh perspective on the biogenesis of type-IV pili.