Natural killer (NK) T lymphocytes are a subpopulation of T lymphocytes regarded as early regulators of immune responses. The majority of NKT cells are restricted by the CD1d molecule. NKT cells have mostly been studied in one single mouse strain, C57BL/6 (B6), because of the absence of NK1.1 in other common mouse strains, and the lack of other reliable surface markers for CD1d-restricted cells. To investigate NKT cell subsets in a mouse strain of a genetic background different from B6, we have back-crossed the NKT cell marker NK1.1 from the B6 mouse to the BALB/c mouse strain. We show that NKT cells in the congenic BALB.B6-NK1.1b mouse share many characteristics with their B6 counterparts, but seem to be deficient in the functional NKT cell subtype characterized by low interleukin-4 and high interferon-γ production, and surface expression of CD49b but not CD69. Moreover, in the thymus but not the spleen of BALB.B6-NK1.1b mice we find a novel Vα14-Jα18 invariant NKT cell subset which is devoid of a set of NK markers, suggesting that these cells represent a less differentiated NKT cell stage, and carries high levels of the T-cell receptor and uses a skewed T-cell receptor Vβ-repertoire.