The amino-terminal amino acid sequences of the pili proteins from four antigenically dissimilar strains of Neisseria gonorrhoeae, from Neisseria meningiditis, and from Escherichia coli were determined. Although antibodies raised to the pili protein from a given strain of gonococcus cross-reacted poorly or not at all with each of the other strains tested, the amino-terminal sequences were all identical. The meningococcal protein sequence was also identical with the gonococcal sequence through 29 residues, and this sequence was highly homologous to the sequence of the pili protein of Moraxella nonliquifaciens determined by other workers. However, the sequence of the pili protein from E. coli showed no similarity to the other sequences. The gonococcal and meningococcal proteins have an unusual amino acid at the amino termini, N-methylphenylalanine. In addition, the first 24 residues of these proteins have only two hydrophilic residues (at positions 2 and 5) with the rest being predominantly aliphatic hydrophobic amino acids. The preservation of this highly unusual sequence among five antigenically dissimilar Neisseria pili proteins implies a role for the amino-terminal structure in pilus function. The amino terminus may be directly or indirectly (through preservation of tertiary structure) important for the pilus function of facilitating attachment of bacteria to human cells.