The mannose-sensitive haemagglutinin (MSHA) pilus that is associated with Vibrio cholerae strains of El Tor biotype has been shown to be a potential colonization factor and protective antigen. The gene encoding the structural subunit of MSHA pili was cloned from size-fractionated SacI-cleaved chromosomal DNA in the expression phage vector lambda ZAPII. Positive clones carried a c. 5.3 kb SacI fragment and were identified on the basis of MSHA expression and hybridization with a synthetic oligonucleotide probe based upon the N-terminus of MshA, the structural subunit of MSHA. The mshA gene was localized to a 2.6 kb SalI-EcoRI fragment, which was subcloned and shown to express MshA from its own promoter in Escherichia coli. Nucleotide sequencing of the entire fragment revealed six open reading frames (ORFs) of which four were complete. The mshA gene encodes an 18,094 Da prepilin protein, which in its mature form has a size of 17,436 Da. MshA is a type 4 (N-MePhe) pilin protein that is more homologous to pilins produced by Pseudomonas aeruginosa and Neisseria gonorrhoeae than to TcpA, the structural subunit of the toxin-coregulated pilus of V. cholerae. The protein seems to be directly involved in receptor binding, as an in-frame mutation in the mshA gene was found to abolish both D-mannose-dependent haemagglutination and binding of V. cholerae bacteria to D-mannose-containing agarose beads. Three additional ORFs, all in the same transcriptional orientation as mshA, were found to encode type 4 pilin-like proteins. A potential promoter with a sequence homologous to that of cAMP-CRP-activated promoters in E. coli was identified upstream of ORF3, the gene preceding mshA.