Aims: Understanding factors that influence the composition of microbial populations of the digestive system of dairy cattle will be key in regulating these populations to improve animal performance. Although rumen microbes are well studied, little is known of the dynamics and role of microbial populations in the small intestine of cows. Comparisons of fingerprints of microbial populations were used to investigate the effects of gastrointestinal (GI) segment and animal on community structure. Methods and Results: Samples from four lactating dairy cows with ruminal, duodenal and ileal cannulae were collected. Terminal-restriction fragment length polymorphism (T-RFLP) comparisons of small subunit rRNA genes revealed differences in microbial populations between GI segments (P < 0 center dot 05). No significant differences in either methanogen populations or microbial community profiles between animals were observed. Quantitative PCR was used to assay relative changes in methanogen numbers compared to procaryote rRNA gene numbers, and direct microscopic counts were used to enumerate total procaryote numbers of the duodenal and ileal samples. Conclusions: T-RFLP comparisons illustrate significant changes in microbial diversity as digesta passes from one segment to another. Direct counts indicate that microbial numbers are reduced by eight orders of magnitude from the rumen, through the abomasum, and into the duodenum (from c. 1012 to c. 3 center dot 6 x 104 cells per ml). Quantitative PCR analyses of rRNA genes indicate that methanogens are present in the duodenum and ileum. Significance and Impact of the Study: The contribution of microbial populations of the small intestine to the nutrition and health of cattle is seldom addressed but warrants further investigation.