V(alpha)14(+) T cells are a unique subset expressing an invariant T-cell antigen receptor ac chain encoded by V(alpha)14 and J(alpha)281 gene fragments with a 1-nt N region. Most infariant V(alpha)14(+) T cells develop in extrathymic organs, independent of thymus, and expand at a high frequency in various mouse strains regardless of major histocompatibility complex (MHC) haplotype. In this paper, we show that the positive selection of invariant V(alpha)14(+) T cells requires a beta(2)-microglobulin-associated MHC class I-like molecule not linked to the MHC on chromosome 17. This was determined by linkage analysis on DNA from recombinant mice generated by crossing a C57BL/6 mouse with a wild mouse, Mus musculus molossinus, that is negative for invariant V(alpha)14 TCR expression, However, the peptide transporter TAP1 is not necessary for positive selection of invariant V(alpha)14(+) T cells, indicating the direct recognition of the MHC class I-like molecule without peptide by the invariant V(alpha)14 TCR Further, experiments with bone marrow-chimeric mice show that invariant V(alpha)14(+) T cells in the periphery are selected by bone marrow cells, suggesting a unique lineage of V(alpha)14(+) T cells differentiated through a selection process distinct from that of conventional alpha beta TCR(+) T cell.