In MHC class II-/- C57BL/6 (II-/-) mouse spleen, a small population of CD4+ T cells is present of which NK1.1+ CD4+ (NK) T cells comprise 40 to 45%. We report here that many of the NK1.1- CD4+ T cells derived from II-/- mice are also NK T cells. They produce large amounts of IL-4 in response to anti-CD3 ligation and do so without any requirement for the presence of IL-4 in the priming culture, a property characteristic of NK T cells. Their IFN-gamma production is large and is enhanced by IL-12. In addition, II-/- NK1.1- CD4+ T cells produce IL-4 as a result of culture with L cells expressing murine CD1 (L-CD1). We report that CD49b, a component of integrin VLA-2, is expressed on the majority of both NK1.1+ and NK1.1- NK T cells. NK1.1- NK T cells also exist in wild-type C57BL/6 mice. Evidence supporting this is that Vbeta8 usage by CD62Llow NK1.1- CD4+ T cells was approximately 5% higher than that by CD62Lhigh CD4+ T cells in wild-type mice in keeping with the estimated proportion of NK1.1- NK T cells in the CD62Llow population. CD62Llow CD4+ T cells from beta2-m(-/-) mice, which lack NK T cells, showed no increase in Vbeta8 usage. When activated by anti-CD3 or L-CD1, CD62Llow NK1.1- CD4+ T cells from conventional but not beta2-m(-/-) and CD1-/- mice produce IL-4 in a manner indistinguishable from II-/- NK1.1- CD4+ T cells. NK1.1- NK T cells in normal mouse spleens are approximately as numerous as NK1.1+ NK T cells.