BACKGROUND:We previously demonstrated the existence of two distinct subsets of T cell receptor (TCR)alphabeta+CD8alphabeta+ single positive (SP) cells in human postnatal thymus which express the chemokine receptor CCR7 or CXCR3 and migrate in vitro in response to their specific ligands. AIM:To investigate whether these two CD8+ thymocyte subsets had distinct peripheral colonisation. METHODS:TCRalphabeta+CD8+ SP cells were obtained from normal postnatal thymus, mesenteric lymph node (LNs), small bowel, and peripheral blood (PB) specimens. Cells were then evaluated for expression of surface molecules, cytolytic potential, telomere length, and profile of cytokine production. RESULTS:CD8+CCR7+CXCR3- thymocytes exhibited CD62L, in common with those which localise to LNs. In contrast, CD8+CCR7-CXCR3+ thymocytes lacked CD62L but exhibited CD103, similar to intraepithelial lymphocytes (IELs) present in the gut mucosa where the CXCR3 ligand, CXCL10, and the CD103 ligand, E-cadherin, are highly and consistently expressed. In addition, thymocytes and gut CD8+CXCR3+CD103+ cells showed comparable telomere length, which was higher than that of PB CXCR3+CD8+ T cells. However, both of these populations contained perforin and granzyme A, and displayed the ability to produce interferon gamma and interleukin 2. Of note, CXCR3 deficient, in comparison with wild-type C57Black/6, mice showed decreased proportions of CD3+CD8alphabeta+ and increased proportions of CD3+CD8alphaalpha+ lymphocytes at gut level. Moreover, adoptive transfer of CD3+CD8alphabeta+ thymocytes from wild-type into CXCR3 deficient mice resulted in a significant increase in CD3+CD8alphabeta+ T cells in the gut mucosa but not in other tissues. CONCLUSIONS:The results of this study demonstrate the existence of a previously unrecognised subset of TCRalphabeta+CD8alphabeta+ SP CXCR3+CD103+ thymocytes which share phenotypic and functional features with CD8+ IELs, thus suggesting the possibility of their direct colonisation of the gut mucosa.