Pathogenic bacteria assemble a variety of adhesive structures on their surface for attachment to host cells. Some of these structures are quite complex. For example, the hair-like organelles known as pili or fimbriae are generally composed of several components and often exhibit composite morphologies. In Gram-negative bacteria assembly of pili requires that the subunits cross the cytoplasmic membrane, fold correctly in the periplasm, target to the outer membrane, assemble into an ordered structure, and cross the outer membrane to the cell surface. Thus, pilus biogenesis provides a model for a number of basic biological problems including protein folding, trafficking, secretion, and the ordered assembly of proteins into complex structures. P pilus biogenesis represents one of the best understood pilus systems. P pili are produced by 80-90% of all pyelonephritic Escherichia coli and are a major virulence determinant for urinary tract infections. Two specialized assembly factors known as the periplasmic chaperone and outer membrane usher are required for P pilus assembly. A chaperone/usher pathway is now known to be required for the biogenesis of more than 30 different adhesive structures in diverse Gram-negative pathogenic bacteria. Elucidation of the chaperone/usher pathway was brought about through a powerful combination of molecular, biochemical, and biophysical techniques. This review discusses these approaches as they relate to pilus assembly, with an emphasis on newer techniques. (C) 2000 Academic Press.