BACKGROUND: We have recently reported the presence of CD8(+) and CD4/8 double-negative (DN) natural killer T (NKT) lymphocytes in sooty mangabeys. To investigate differences in the two NKT cell subsets, we compared the phenotype and function of sooty mangabey CD8(+) and DN NKT cells. METHODS: Flow-sorted NKT lymphocytes from one SIV-negative sooty mangabey were subjected to limiting dilution cloning. Invariant NKT clones were characterized by flow cytometry and cytokine ELISA. RESULTS: The majority of NKT clones displayed an effector memory phenotype and expressed CXCR3 and NKG2D. While CD8(+) NKT subsets expressed significantly higher levels of granzyme B and perforin and produced more IFN-gamma, the DN NKT subsets secreted significantly more IL-4, IL-13, and IL-10. CONCLUSIONS: The Th1 and Th2 cytokine bias of CD8(+) and DN NKT cells, respectively, indicates the presence of functionally heterogeneous populations of NKT cells in sooty mangabeys.