Escherichia coli strain RDEC-1 (serotype 015:NM) is an effacing adherent enteropathogen that binds to the intestine of rabbits in a manner morphologically identical to the binding of human enteropathogenic E. coli strains to human intestine. The rabbit enteropathogen adheres to mucosal enterocytes in vivo and to microvillus membranes in vitro. Binding of RDEC-1 to ileal brush borders and to M cells overlying Peyer's patches is mediated by pili (fimbriae) expressed on the cell surface of bacteria. To examine whether similar binding occurs to glycoproteins present in the intestinal lumen, RDEC-1 was fed to rabbits and the intestinal luminal contents were examined for in vivo colonization by RDEC-1. In addition, preparations of rabbit luminal glycoproteins were tested for their ability to agglutinate RDEC-1 in vitro. After the infection of rabbits, RDEC-1 organisms were found in the intestinal lumen associated with glycoproteins, as defined by positive histochemical staining of luminal material by periodic acid-Schiff and Alcian blue. In vitro aggregation of RDEC-1 by luminal glycoproteins of rabbit intestine, a luminal glycoprotein fraction purified on column chromatography and rabbit ileal microvillus membranes, was determined using a microtiter plate assay and an aggregometer. Nonpiliated RDEC-1 did not interact with either of the intestinal glycoprotein preparations or microvillus membranes. RDEC-1 expressing mannose-resistant AF/R1 pili or mannose-sensitive type 1 pili coaggregated with both rabbit luminal glycoprotein preparations and rabbit ileal microvillus membranes at equivalent protein concentrations. These studies demonstrate that RDEC-1 are associated with luminal glycoproteins during in vivo infection of rabbits, and that piliated RDEC-1, but not nonpiliated, coaggregate with rabbit glycoprotein preparations in vitro. Luminal glycoproteins contained within the mucus layer may serve as a site for replication and colonization of bacteria before bacterial enteroadherence.