Monoclonal antibody YK-3 was established by immunization with IV3 alpha (NeuGc alpha 2-8NeuGc)-Gg4Cer (GD1c-(NeuGc-NeuGc-)), and its epitope was determined to be NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1. Thin layer chromatography immunostaining with YK-3 detected only GD1c-(NeuGc-NeuGc-) among the gangliosides of mouse thymocytes and splenocytes. Immunohistochemical staining with YK-3 visualized the medulla of mouse thymus and T cell-dependent areas of mouse spleen and mesenteric lymph nodes. Two-color flow cytometry demonstrated that GD1c (NeuGc-NeuGc-) was expressed on a quarter of CD3+ mature thymocytes and strongly expressed on three quarters of CD4+ T cells in the spleen, lymph nodes, and peripheral blood but not on CD8+ T cells or B cells. GD1c (NeuGc-NeuGc-)-positive cells and negative cells were separated by panning with YK-3 on Petri dishes into adherent and nonadherent fractions. Following stimulation with concanavalin A, adherent cells, predominantly GD1c (NeuGc-NeuGc-)+, produced more interleukin-2 (IL-2) and markedly less interleukin-4 (IL-4) than nonadherent cells. This conclusion is supported by data obtained by lysis of cells by YK-3 and complement. These data indicate that the cell surface expression of GD1c (NeuGc-NeuGc-) is restricted to a small number of mature thymocytes and a subset of CD4+ T cells, which produce abundant IL-2 and very little IL-4, suggesting that GD1c(NeuGc-NeuGc-) is an excellent marker for mouse naive T or T helper 1-like cells in vivo.