Recent studies have increased our understanding on the mechanistic basis of the proposed probiotic health effects. Well designed human studies have demonstrated that specific probiotic strains have health benefits in the human population. These have led to a wide acceptation of the probiotic concept. However, current probiotics have not been selected for specific purposes. Novel methods to select and characterise target-specific probiotic strains are thus needed. In addition to the traditional selection procedures, in recent years, knowledge on intestinal microbiota, nutrition, immunity and mechanisms of action has increased dramatically and can now be combined with genomic data to allow the isolation and characterization of new target- or site-specific probiotics. We should expect to see new, third generation probiotics emerging in the near future and also new selection criteria further defining the targets of future probiotics.