The gene encoding the K99 fibrillar adhesin of Escherichia coli has been modified by oligonucleotide-directed, site-specific, mutagenesis. The tryptophan-67, lysine-132, lysine-133 or arginine-136 were replaced by leucine, threonine, threonine and serine, respectively. The threonine-133 mutant fibrillae were indistinguishable from wild-type fibrillae. In contrast, replacement of lysine-132 or arginine-136 by threonine or serine, respectively, resulted in mutant fibrillae which had completely lost adhesive capacity, suggesting that the positive charges of these residues are essential for the interaction with the negatively charged sialic acid residue of the receptor molecules. After the replacement of tryptophan-67 with leucine neither fibrillae nor subunits were detectable, indicating that the mutant product is unstable and that tryptophan-67 has an essential structural role in the K99 subunit.