Enteroaggregative Escherichia coli (EAggEC) are diarrheal pathogens defined by aggregative adherence to HEp-2 cells. In an effort to identify pathogenic EAggEC isolates, four groups of 5 volunteers were fed 1 of 4 different EAggEC strains, each at a dose of 1010 cfu. Strain 042 caused diarrhea in 3 of 5 adults; 3 other EAggEC isolates (17–2, 34b, and JM221) failed to elicit diarrhea. A gene encoding enterotoxin EASTI was found in strains 042 and 17–2 but not 34b or JM221; a 108-kDa cytotoxin was expressed in all 4 isolates. All 4 isolates showed a modest degree of gentamicin protection in HEp-2 cells. 17–2, 34b, and JM221 expressed the fimbrial antigen AAF/I; 042 did not express this fimbria as determined by immunogold electron microscopy and genetic probe hybridization.