We have used two monoclonal anti-murine T cell autoantibodies (SM3G11 and SM6C10) and multi-color immunofluorescence staining to resolve splenic CD4+ cells into four populations. Two of these populations (Fr. I and Fr. III, 35% and 10% of CD4+ cells) show mutually exclusive expression of these determinants and exhibit distinct functions. Fr. III secretes IL-4, but not IL-2 when activated by Con A, and includes memory T cells responsible for secondary antibody formation. In contrast, Fr. I secretes IL-2 but not IL-4 in response to Con A, and does not contribute to the secondary antibody response. Furthermore, these two fractions exhibit differential accessory cell dependence. Whereas Fr. III responds with B cells (and also non-B cells) as accessory cells in Con A-induced activation, Fr. I requires non-B cells. However, we found that many CD4+ cells (Fr. II, 40% of CD4+ cells) express both determinants and are not distinguishable with regard to lymphokine secretion, accessory cell effect, and memory T cell activity. Curiously, the fraction expressing neither determinant (Fr. IV, 10% of CD4+ cells) is unresponsive to experimental conditions used here. We discuss the possible relationships between these T cell subsets and the implications of differential expression of these determinants.