CD161 (NKR-P1) is a natural killer receptor of C-type lectin superfamily found on natural killer (NK) cells. A significant number of both human CD4+ and CD8+ T cells express CD161. However, little is known about the extended phenotype or function of CD161+ T cells. Here, we analyzed this population in normal human peripheral blood by immunofluorescent staining, and multi-color flow cytometric analysis (N=15). Amongst CD4+ T cells, a mean of 22% showed intermediate staining for CD161 (CD161int) and the remainder were CD161−. Almost all the CD4+CD161int TCRαβ+ T cells had the CD45RO+ memory phenotype, and did not express the CD16 or CD56 NK markers (<5%). CD4+CD161− T cells were a mixture of CD45RO+ and CD45RA+ cells. Invariant natural killer T (NKT) cells with the Vα24+/Vβ11+ TCR are known to express CD161, but CD4+CD161int T cells contained less than 1 % of these invariant NKT cells. After in vitro stimulation with αCD3 and αCD28 mAb, CD4+CD161int T cells produced larger amounts of both Th1 and Th2 type cytokines compared with CD4+CD161− T cells, especially IFN-γ, IL-4 and IL-10. However, there was no difference in the proliferative response between CD161− and CD161int CD4+ T cells, and CD4+CD161int T cells had no suppressor functions against autologous cell proliferation. In CD8+ T cells, there were two populations that were CD161 positive. One was CD161hi (mean; 11%) and another was CD161int (mean; 9%). Almost all CD161int and CD161hi CD8+ T cells had the CD45RO+ memory phenotype, and few expressed the Vα24+/Vβ11+ TCR NKT cell marker (<1%) or CD16 (<5%). CD8+CD161hi cells contained around 15% of CD56+ and CD8+CD161int cells contained around 5% of CD56+. CD8+CD161hi T cells had decreased expression of CD8β and 40% of CD8+CD161hi T cells expressed only CD8α. Conversely, most of the CD8α+CD8β− T cells in human peripheral blood expressed CD161. After in vitro stimulation with αCD3 and αCD28 mAb, CD8+CD161hi T cells secreted no IFN-γ, TNF-α or IL-2. Both CD8+CD161− and CD8+CD161int T cells produced all three cytokines. CD8+CD161hi T cells failed to proliferate after αCD3+αCD28 stimulation, though CD8+CD161− and CD8+CD161int T cells made vigorous proliferative responses. Thus, it appeared that CD8+CD161hi T cells were anergic. This anergic state could not be reversed by addition of IL-2. CD8+CD161+ T cells had no suppressor function against autologous cell proliferation nor cytotoxicity against the NK or NKT sensitive tumor cell lines, K562 and Jurkat. In conclusion, the CD161 marker can be used to identify subsets of CD4+ and CD8+ T cells that differ in their extended phenotypes and functions. In addition, we identified a unique subset of anergic CD8+CD161hi T cells that express a memory phenotype with a low level of CD8β.