Pathogenic bacteria frequently possess pili with specific binding properties that allow them to attach to epithelial tissue. In Escherichia coli, the pili associated with pyelonephritis (Pap pili) bind to digalactoside-containing glycolipids on the uroepithelium. Transposon-insertion mutants and deletion mutants of the cloned genetic determinant encoding synthesis of such digalactoside-binding Pap pili have been studied in E. coli K-12. Mutants that completely lack synthesis of the major Pap pili subunit protein, the papA gene product, and thereby no longer produce pili were shown to retain the binding specificity of intact Pap pili. Reduced expression of some of the remaining pap genes, presumably due to polarity effects from papA::Tn5 insertions, was circumvented by the use of a copy-number mutant plasmid vector. Derivatives carrying the papA-D genes produced Pap pili but did not bind to human cells. The products of the genes papE-G are essential for digalactoside-specific hemagglutination and for attachment to urinary bladder cells. The papC and papD genes presumably aid in surface localization and/or polymerization of the pili-adhesin subunits and are required for expression of pili as well as of the binding properties. Serological evidence is presented that suggests that a minor pilus component(s), presumably produced by the papE, -F, or -G gene, is the actual binding moiety in the digalactoside-specific interaction of Pap pilus-adhesin.