Background: Natural killer T cells represent a linkage between innate and adaptive immunity. They are a heterogeneous population of specialized T lymphocytes composed of different subsets. DX5(+)NKT cells are characterized by expression of the NK cell marker DX5 in the context of CD3. However, little is known about the phenotype and functional capacity of this unique cell population. Therefore, we investigated the expression of several T cell and NK cell markers, as well as functional parameters in spleen and liver subsets of DX5(+)NKT cells in NK1.1(-) Balb/c mice and compared our findings to NK1.1(+) C57Bl/6 mice. Results: In the spleen 34% of DX5(+)NKT cells expressed CD62L and they up-regulated the functional receptors CD154 as well as CD178 upon activation. In contrast, only a few liver DX5(+)NKT cells expressed CD62L, and they did not up-regulate CD154 upon activation. A further difference between spleen and liver subsets was observed in cytokine production. Spleen DX5(+)NKT cells produced more Th1 cytokines including IL-2, IFN-gamma and TNF-alpha, while liver DX5(+)NKT cells secreted more Th2 cytokines (e. g. IL-4) and even the Th17 cytokine, IL-17a. Furthermore, we found inter-strain differences. In NK1.1(+) C57Bl/6 mice DX5(+)NKT cells represented a distinct T cell population expressing less CD4 and more CD8. Accordingly, these cells showed a CD178 and Th2-type functional capacity upon activation. Conclusion: These results show that DX5(+)NKT cells are a heterogeneous population, depending on the dedicated organ and mouse strain, that has diverse functional capacity.