Thymocytes expressing homogeneous TCR V gamma 5/V delta 1 gene without N nucleotide addition (canonical sequence) have been reported to be positively selected in fetal thymus. Because skin intraepithelial lymphocytes (s-IEL) also express canonical sequence, it is proposed that s-IEL are generated by an epidermis-specific homing of these positive selected fetal V gamma 5/V delta 1-bearing thymocytes. We have reported that positive selection of V gamma 5+ thymocytes is not seen in AKR/J-->C3H/He bone marrow chimeras. In this report, we analyzed the phenotype of donor-derived s-IEL from AKR/J-->C3H/He bone marrow chimeras that appeared in the absence of positive selection of V gamma 5-bearing thymocytes. We found interestingly that almost all donor-derived s-IEL in the bone marrow chimeras expressed TCR-alpha beta lack expression of both CD4 and CD8. Furthermore, TCR-alpha beta+ s-IEL were detected in adult-thymectomized bone marrow chimeras, indicating that the TCR-alpha beta+ s-IEL are extrathymically derived. All these results suggest that the s-IEL expressing TCR-alpha beta appear extrathymically in the absence of positive selection of V gamma 5/V delta 1-bearing thymocytes. Our results may also explain the contradicting result between human s-IEL, which are TCR-alpha beta+ CD4- CD8- and murine s-IEL, and suggest the importance of extrathymic T cell differentiation pathway of the TCR-alpha beta lineage in immune surveillance.