To test the hypothesis that establishing gastrointestinal colonization with multiresistant Enterococcus faecium (VRE) C68 results from expansion of the enterococcal population in the upper small bowel, we compared VRE quantities recovered from the proximal, middle, and distal segments of the small bowel from mice treated with different antimicrobial agents. Antibiotics associated with high-level VRE fecal colonization (cefotetan, ceftriaxone, clindamycin, and ticarcillin-clavulanic acid) increased VRE quantities in all small-bowel segments, whereas cefepime and piperacillin-tazobactam did not. Enterococcal expansion did not correlate with reductions in numbers of native gram-negative or anaerobic flora. Green fluorescence protein-expressing E. faecium bacteria were found adjacent to the small bowel epithelial lining in colonized mice. These data indicate that enterococcal bowel colonization begins within the proximal small bowel and does not correlate with inhibition of other cultivable flora. Host or enterococcal factors induced by exposures to certain antibiotics may play a role in facilitating E. faecium colonization of the mammalian gastrointestinal tract.