A population of human T cells expressing an invariant V 24J QT cell antigen receptor (TCR) chain and high levels of CD161 (NKR-P1A) appears to play an immunoregulatory role through production of both T helper (Th) type 1 and Th2 cytokines. Unlike other CD161 T cells, the major histocompatibility complex–like nonpolymorphic CD1d molecule is the target for the TCR expressed by these T cells (V 24invt T cells) and by the homologous murine NK1 (NKR-P1C) T cell population. In this report, CD161 was shown to act as a specific costimulatory molecule for TCR-mediated proliferation and cytokine secretion by V 24invt T cells. However, in contrast to results in the mouse, ligation of CD161 in the absence of TCR stimulation did not result in V 24invt T cell activation, and costimulation through CD161 did not cause polarization of the cytokine secretion pattern. CD161 monoclonal antibodies specifically inhibited V 24invt T cell proliferation and cytokine secretion in response to CD1d target cells, demonstrating a physiological accessory molecule function for CD161. However, CD1d-restricted target cell lysis by activated V 24invt T cells, which involved a granule-mediated exocytotic mechanism, was CD161-independent. In further contrast to the mouse, the signaling pathway involved in V 24invt T cell costimulation through CD161 did not appear to involve stable association with tyrosine kinase p56Lck. These results demonstrate a role for CD161 as a novel costimulatory molecule for TCR-mediated recognition of CD1d by human V 24invt T cells.