CD1d-deficient (CD1d^−/−) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a Vα19.1–Jα26 (AV19–AJ33) invariant TCR α chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.1⁺ but not NK1.1⁻ T cell population isolated from CD1d^−/− mice. Moreover, more than half (11/21) of the hybrid cell lines established from CD1d^−/− NKT cells expressed the Vα19.1–Jα26 invariant TCR α chain. The expression of the invariant Vα19.1–Jα26 mRNA was absent in β2-microglobulin-deficient mice. Collectively, the present findings suggest the presence of a second NKT cell repertoire characterized by an invariant TCR α chain (Vα19.1–Jα26) that is selected by an MHC class I-like molecule other than CD1d.