CD1d-restricted Vα14-Jα281 invariant αβTCR+(NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1. 1-expressing strains. Surrogate markers for NKT cells such as αβTCR+ CD4− CD8− and DX5+ CD3+ have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1. 1-congenic BALB/c, and NK1. 1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1. 1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/α-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5+ CD3+) actually excludes these cells. Finally, our results support the concept that NK1. 1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1. 1-negative, especially within the CD4+ subset and particularly in NK1. 1-congenic BALB/c mice.