During the past decade it became evident that anaerobic cultivation-based approaches provides an incomplete picture of the microbial diversity in the GI tract, since at present only a minority of microbes can be obtained in culture. The application of molecular, mainly 16S ribosomal RNA (rRNA)-based approaches enables researchers to bypass the cultivation step and has proven its usefulness in studying the microbial composition in a variety of ecosystems, including the gastrointestinal GI tract. This critical review summarizes the impact of these culture-independent approaches on our knowledge of the ecology of the GI tract and provides directions for future studies which should emphasize function of specific strains, species and groups of microbes.