The afa-3 gene cluster determines the formation of an afimbrial adhesive sheath that is expressed by uropathogenic as well as diarrhoea-associated Escherichia coli strains. It contains six genes (afaA-afaF), among which the afaE3 gene is known to code for the structural AfaE-III adhesin (previously designated AFA-III), whereas no role has yet been identified for the afaD gene product. The afa-3 gene cluster is closely related to the daa operon that codes for an adhesin, the F1845 adhesin, which is highly related to the AfaE-III adhesin; however, unlike the AfaE-III adhesin, F1845 is a fimbrial adhesin. Reported in this work is the construction of chimeras between the afa-3 and daa operons. Analyses of the phenotypes conferred by these afa-3/daa chimeric clusters allowed us to conclude that the biogenesis of a fimbrial or an afimbrial adhesin is fully determined by the amino acid sequence of the AfaE-III and F1845 adhesins. Moreover, the role of the AfaD product in the biosynthesis of the afimbrial sheath was assessed by immunogold and immunofluorescence experiments. The AfaD and the AfaE-III products were purified and used to raise rabbit and mouse antisera. Similar to AfaE-III, AfaD was found to be a surface-exposed protein as well as an adhesin; both AfaD and AfaE-III are concomittantly expressed by the bacterial cell. These results demonstrate, for the first time, that the afimbrial adhesive sheath expressed by pathogenic E. coli is composed of two adhesins.