The adherence of enterotoxigenic Escherichia coli (ETEC) to the human small intestine is an important early event in infection. Attachment is thought to be mediated by proteinaceous structures called pili. We have investigated the regulation of expression of the genes encoding CS1 pili found on human ETEC strains and find that there are at least three promoters, P1 and P2, upstream of the coo genes, and P3, downstream of the start of cooB translation. We identified a silencer of transcription which extends over several hundred bases overlapping the cooB open reading frame. This silencer is dependent on the promoter and/or upstream region for its negative effect. The DNA binding protein H-NS is a repressor of coo transcription that acts in the same region as the silencer, so it is possible that H-NS is involved in this silencing. Rns, a member of the AraC family, positively regulates transcription of the coo operon and relieves the silencing of CS1 expression.