Bacteria belonging to the genus Lactobacillus are members of the lactic acid bacteria (LAB), a broadly defined group characterized by the formation of lactic acid as the sole or main end product of carbohydrate metabolism. They can be found in plants or material of plant origin, silage, fermented food (yogurt, cheese, olives, pickles, salami, etc.), as well as in the oral cavities, gastrointestinal tracts (GIT), and vaginas of humans and animals (31). In particular, the Lactobacillus species found in the GIT have received tremendous attention due to their health-promoting properties. They are commonly used as probiotics, which are defined by the FAO/WHO as live microorganisms that when administered in adequate amounts confer a health benefit on the host. The economic success and exciting prospects of probiotic products have accelerated research on intestinal lactobacilli. Genomics of Lactobacillus species is booming, and the genomes of five strains that belong to species commonly found in human fecal samples have recently been sequenced (50). Several comparative and functional genomic investigations have been conducted to gain information about the functionality of lactobacilli in the GIT (69). Unfortunately, a major misconception regarding the ecological role of lactobacilli in the intestinal tract has been embraced by many scientists working in the field. Specifically, there has been a general and persistent assumption that a large number of Lactobacillus species form stable and numerically significant populations in the human intestinal tract, especially in the small intestine, where they are presumed to form epithelial associations (101). Considering how widespread and accepted this perception is, there is surprisingly little experimental evidence that supports it. Ecological observations for the prevalence and dynamics of fecal Lactobacillus populations and the findings obtained with comparative genomics do indicate now that the ecological role of most types of intestinal lactobacilli, and their relationship with the human host, should be reconsidered. In this review, evidence is summarized that suggests that only a small number of Lactobacillus species are true inhabitants of the mammalian intestinal tract and that most lactobacilli present are allochthonous members derived from fermented food, the oral cavity, or more proximal parts of the GIT. It is further explained why this knowledge provides information valuable for selecting strains for fundamental research of the ecological role of lactobacilli in the GIT, for their use as probiotics in foods and supplements, and for pharmaceutical applications.