Natural killer T (NKT) lymphocytes express an invariant T cell antigen receptor (TCR) encoded by the V(alpha)14 and J(alpha)281 gene segments. A glycosylceramide-containing alpha-anomeric sugar with a longer fatty acyl chain (C-26) and sphingosine base (C-18) was identified as a ligand for this TCR. Glycosylceramide-mediated proliferative responses of V(alpha)14 NKT cells were abrogated by treatment with chloroquine-concanamycin A or by monoclonal antibodies against CD1dN(beta)8, CD40/CD40L, or B7/CTLA-4/CD28, but not by interference with the function of a transporter-associated protein. Thus, this lymphocyte shares distinct recognition systems with either T or NK cells.