A hybridoma, 25T3 (IgM, kappa), was established from MRL/+ mice immunized with an autoreactive T cell line (l/+T1). The antigenicity of the antigen recognized by hybridoma 25T3 (25T3-Ag) expressed on thymic and splenic cells was abolished by treatment with phosphatidylinositol-specific phospholipase C, showing that 25T3-Ag is a glycophosphatidylinositol-anchored Ag. 25T3-Ag was expressed on approximately 90% of thymocytes. Double-negative, double-positive and CD8 single-positive cells were highly positive for the expression of 25T3-Ag, whereas CD4 single-positive cells were weakly positive (approximately 40%) or negative (approximately 60%). In the spleen, only CD3+ cells (and not B220+ nor Mac-1+ cells) reacted with 25T3 monoclonal antibody (mAb), indicating that 25T3 mAb is specific for T cells. The majority of splenic CD8+ T cells were positive for the expression of 25T3-Ag, although the intensity was weaker than that of thymocytes. In contrast, splenic CD4+ T cells were divided into negative (60-70%) and positive (30-40%) populations. Similar staining profiles were observed in BALB/c, C57BL/6, C3H/HeN and AKR/J mice. When BALB/c CD4+ T cell subsets were sorted and cultured with irradiated (25 Gy) antigen-presenting cells, stimulation with immobilized anti-CD3 mAb for 2 days resulted in CD4+25T3+ cells secreting more interleukin-2 and less interleukin-4 than did CD4+25T3- subsets, although the proliferative responses of the cells on day 2 of culture were similar. This suggests that CD4+ T cells can be divided into two populations and relatively defined as T helper 1 and T helper 2 cells using this 25T3 mAb. Immunoprecipitation and SDS-PAGE revealed that 25T3-Ag was approximately 70 kDa. These findings are discussed in relation to CD4+ T cell subsets.