We investigated the TCRβ repertoire and the functional properties of MR1-restricted mucosal-associated invariant T (MAIT) cells in healthy individuals. By combined single cell cloning and RNA deep sequencing we found that human MAIT cells are extremely oligoclonal in both blood and liver, display high inter-individual homology, and exhibit a restricted length of the CDR3β domain. The analysis of CDR3β clonotypes distribution at two different time points in three healthy donors also showed that the MAIT cell repertoire is remarkably stable in human blood. We identified and cloned a second oligoclonal sub-population of MAIT cells expressing an alternative invariant TCRα rearrangement conserved between individuals and reacting to riboflavin-producing microbes in MR1-restricted manner. Both MAIT cell subsets were detected at high frequencies in blood and displayed a marked tropism for different non-lymphoid solid organs. Upon bacterial recognition they released large amounts of soluble mediators, including Th0, Th1 and Th2 cytokines and sCD40L, showed cytotoxic capacity against infected cells and promoted killing of intracellular bacteria. In addition, ex vivo multiparametric mass cytometry analysis revealed that different functional subpopulations exist among circulating MAIT cells. All together, these results indicate that human MAIT cells, although recognizing conserved antigens and being composed of relatively few expanded clones, are functionally heterogeneous, thus highlighting important protective and immunoregulatory functions of these lymphocytes and suggesting their contribution not only to infectious diseases but also to immunomediated inflammatory pathologies.