PURPOSE:The aim of this study was to standardize the methods of sample collection of mucus from the digestive tract and to determine the microbiota in healthy volunteers from Brazil, collecting samples from the mouth, esophagus, stomach, duodenum, jejunum, ileum, colon, and rectum. METHODS: Microbiota of selected healthy volunteers from the oral cavity (n=10), the esophagus (n=10), the upper digestive tract (n=20), and the lower digestive tract (n=24) were evaluated through distinct collection methods. Collection methods took into account the different sites, using basic scraping and swabbing techniques, stimulated saliva from the oral cavity, irrigationaspiration with sterile catheters especially designed for the esophagus, a probe especially designed for upper digestive tract, and a special catheter for the lower digestive tract. RESULTS: (i) Mixed microbiota were identified in the oral cavity, predominantly Gram-positive aerobic and anaerobic cocci; (ii) transitional flora mainly in the esophagus; (iii) Veillonella sp, Lactobacillus sp, and Clostridium sp in the stomach and duodenum; (iv) in the jejunum and upper ileum, we observed Bacteroides sp, Proteus sp, and Staphylococcus sp, in addition to Veillonella sp; (v) in the colon, the presence of "nonpathogenic" anaerobic bacteria Veillonella sp (average 10 5 UFC) indicates the existence of a low oxidation-reduction potential environment, which suggests the possibility of adoption of these bacteria as biological markers of total digestive tract health. CONCLUSIONS: The collection methods were efficient in obtaining adequate samples from each segment of the total digestive tract to reveal the normal microbiota. These procedures are safe and easily reproducible for microbiological studies.