Escherichia coli (RDEC-1) adheres to Peyer's patch and absorptive epithelium in the rabbit in the closely adhering manner characteristic of enteropathogenic and enterohemorrhagic E. coli. An adherence pilus (AF/R1) is important for adherence to Peyer's patch M cells in vivo and ileal brush borders in vitro. A nonpiliated mutant (42-2-37-8) of the RDEC-1 strain colonized the gut lumen less readily than the parent strain. The mutant adhered infrequently to Peyer's patch lymphoid follicle epithelium (22% vs. 84%, P less than .0001). Ileal close adherence was less frequent at 3 d, but by 9 d after inoculation had increased, approaching that of the parent RDEC-1 strain. However, adherence was focal, and fewer bacteria were present at each adherence site compared with the parent RDEC-1 strain. The result was a lower frequency of diarrhea (34% vs. 65%, P = .003) and mortality (9.4% vs. 27%, P = .035) with the 42-2-37-8 strain. Loss of AF/R1 pili compromised the ability of the RDEC-1 strain to adhere to Peyer's patch and absorptive epithelium and to produce diarrhea.