In this study, we reexamined the structural prerequisites for the attachment of P-fimbriated Escherichia coli to human urinary tract epithelial cells. The epithelial cells were obtained from A1Pj nonsecretor individuals, who express the globoseries of glycolipids without the ABH blood group determinants, and from A1Pj secretor individuals, who in addition express globo-A, a receptor for the prsj96 adhesin. The wild-type E. coli strains J96, ADI110, and L42 and the recombinant clones HB101 papj96, HB101 prsj96, HB101 papx4,, and HB101 papADilO were tested for binding. They expressed P fimbriae, as defined by P blood group-dependent agglutination of human erythrocytes of the globoseries, but differed in reactivity with galactoseal-4galactoseI3 (Galal-4Galjp)-latex beads, isolated glycolipids of the globoseries, sheep erythrocytes, and uroepithelial cells. Three different patterns of binding were represented among the recombinant clones. HB101 papL4, and HB101 papAD1lO agglutinated sheep erythrocytes and Galael-4GalI-latex beads and attached to both secretor and nonsecretor epithelial cells. HB101 prsj96 agglutinated sheep erythrocytes, reacted poorly with Galrl-4Galflatex beads, and attached to A1 secretor but not to A1 nonsecretor epithelial cells. HB101 papjg6 agglutinated Galcl-4GalW1-latex beads but not sheep erythrocytes and attached poorly to human uroepithelial cells. The receptors relevant for adhesion were analyzed by inhibition with glycolipids in suspension. The sheep erythrocyte agglutination and attachment to secretor and nonsecretor epithelial cells of HB101 papI42 and HB11 papADl1O were inhibited by globotetraosylceramide, while the Forssman glycolipid had no effect. The sheep erythrocyte reactivity and attachment to secretor epithelial cells of HB101 prsj96 were inhibited by the Forssman glycolipid. These results permitted three conclusions. First, the expression of functionally active Galal-4Gal3-specific adhesins, as in HB101 papJ96, was not sufficient to make E. coli competent to attach to human uroepithelial cells. Attachment required P fimbriae of the papLA, orpapAD11o type. Second, the sheep erythrocyte reactivity of P-fimbriated strains could not be attributed solely to recognition of the Forssman glycolipid and may not be used to define the prsJ96-encoded phenotype. Third, the P-fimbrial adhesins which mediate secretor state-independent attachment to human uroepithelial cells recognized receptor epitopes provided by globotetraosylceramide.