Gut microbiota has been suggested as a key component of gut homeostasis, affecting immune responses within the gut. We determined changes in intestinal commensal bacteria and expression of toll-like receptors (TLR) 2 and 4 in rats bred under microbiologically controlled conditions (barrier), under standard conditions (conventional), and in barrier animals adapted to standard conditions (barrier/conventional). Cecal microbiota was analyzed by plate culture, and fluorescence in situ hybridization and microbial profiles were assessed by terminal restriction fragment length polymorphism. Cecal expression of TLR-2 and TLR-4 was determined by reverse transcription polymerase chain reaction (PCR). Total number of cecal bacteria was similar in the three groups. However, the barrier group showed a higher number of strict anaerobic bacteria (Bacteroides spp. and Clostridium spp.) while Bifidobacterium spp. were scarce. Re-housing the barrier-bred rats into conventional conditions led to a microbiota with intermediate characteristics between the barrier and conventional groups. Richness of the cecal microbial ecosystem was similar in the three groups, although a relative time-dependent variation, with highest homogeneity in the barrier group, was observed. Expression levels of TLR-2 and TLR-4 had no clear correlation with the microbiota. These results show that the relative composition of the cecal microbiota in rats varies spontaneously with changes in the environmental conditions, with minor impact in the expression of TLR-2 and TLR-4. These observations might be important in the understanding of variability in animal responses, particularly to immune-related stimuli, when assessed in the context of the environmental/microbiological conditions. © 2010 Springer Science+Business Media, LLC.