Broilers are withheld from feed for 8 to 24 h before processing to empty the gastrointestinal tract and reduce potential carcass contamination from gastrointestinal tract contents. Intestinal microbial changes during feed withdrawal (FW) have not been thoroughly defined. Two experiments were conducted to examine the effects of diet and FW on the microbial ecology in the small intestine. In experiment 1, 42-d-old broilers were fed diets containing no additive (control), 250 ppm of CuSO(4), or bacitracin (BMD; 30 ppm) and were also subjected to FW for 0, 10, and 24 h. Six birds from each dietary treatment were killed at each FW time point and ileal mucosa and digesta were collected. Microbial communities were determined by isolating bacterial DNA, amplifying the V3 region of 16S ribosomal DNA, and performing denaturing gradient gel electrophoresis. The mucosal microbial profiles from birds at 0 h of FW had higher similarity values than those at 24 h of FW, indicating that as FW time increased, uniformity of intestinal microbial populations decreased. Numbers of bands (an indicator of numbers of bacterial species present) at 0 h (9.38) were greater than those at 10 and 24 h (5.39 and 5.78, respectively), suggesting a reduction in microbial species and diversity as FW time increased. Copper-fed birds had greater similarity coefficients than either the control or BMD-fed birds, but BMD-fed birds had greater band numbers. No interaction between diet and FW was observed and no digesta differences were observed. In experiment 2, 62-d-old birds fed corn-soy diets in floor pens were subjected to 0, 8, 12, and 24 h of FW. Ileal mucosal tissue was collected and analyzed as in experiment 1. Mucosal microbiota similarities were greater at 0 h of FW than at 8, 12, or 24 h of FW and band numbers were reduced between 0 and 24 h of FW. Data from these studies suggest that FW and dietary treatments alter the microbial community of the intestine by decreasing bacterial diversity in the ileum.