We have previously defined four murine CD4+ peripheral T cell subsets, fractions (Fr.) I-IV, based on expression of the 6C10 and 3G11 determinants (Hayakawa, K. and Hardy, R. R., J. Exp. Med. 1988. 168: 1825). These subsets also show distinctive levels of other cell surface markers: the two minor subsets, Fr. III and Fr. IV, are both CD45RBlow/-, L-selectin (Mel-14)- and CD44hi, characteristic of secondary T cells. The patterns and levels of cytokine production by individual cells in each subset were determined by bioassay for interleukin (IL)-2/IL-4 or IL-4/interferon (IFN)-gamma production after anti-CD3 stimulation. Our data revealed that these four phenotypically defined subsets largely coincide with clusters of cells showing uniform distinctive cytokine profiles, i.e. IL-2+/IFN-gamma-/IL-4- (Fr. I and Fr. II, L-selectin+), IL2+/IFN-gamma +/IL-4+ (Fr. III, L-selectin-), and IL-2-/IFN-gamma low/-/IL-4+ (Fr. IV, L-selectin-). Besides these subsets, an L-selectin-negative cell subfraction within Fr. II appears to represent a transitional population between the IL-2+/IFN-gamma-/IL-4- stage and the IL-2+/IFN-gamma +/IL-4+ stage. Taken together, these results demonstrate the presence of two IL-4+ secondary T cell subsets with distinct cytokine production patterns, and show that the majority of IL-4+ cells found in healthy adult laboratory mice co-produce IFN-gamma, and thus are not typical T helper type 2 cells.