Valpha24 natural killer T (NKT) cells are innate immune cells that recognize self and nonself glycolipids presented by CD1d molecules, and play immunoregulatory roles in autoimmunity and tumor immunity. We have investigated the circulating Valpha24 NKT cells in a large cohort of human subjects. CCR7(-) CD45RO(+) effector memory cells dominated both CD4(+) and CD4() NKT subsets, while a minority displayed a central memory phenotype. CD4(-) central memory NKT cells, however, were atypical in that they largely lacked CD62L expression. Overall, CD4(-) NKT cells displayed a functional phenotype with effector characteristics, while the CD4(+) subset appeared immunoregulatory. Interestingly, NKT cell numbers in blood varied widely between subjects, and elevated numbers of these cells were much more common in women than in men. The CD4(+) subset dominated the NKT cell compartment in both sexes, while circulating NKT cell numbers above 0.1% were associated with an expanded CD4(-) subset. Although NKT cell numbers were generally stable over time, we describe a dynamic fivefold expansion that was associated with a skewing of the NKT CD4(+):CD4(-) ratio that persisted after numbers returned to base line. Thus, the two NKT cell subsets display different properties and dynamics that will influence their function as innate immunoregulatory and effector cells.