CD4+ T helper (Th) cells can be classified into different types based on their cytokine profile. Cells with these polarized patterns of cytokine production have been termed Thl and Th2, and can be distinguished functionally by the production oflFN-γ and IL-4, respectively. These phenotypes are crucial in determining the type of immune response that develops after antigen priming. There are no surface markers that define them, and cytokine immunoassay or mR.NA analysis both have limitations for characterization of single cells. Using immunofluorescent detection of intracellular IFN-γ and IL-4, we have studied the emergence of Thl and Th2 cells in response to antigen exposure and the patterns of cytokine synthesis in established T cell clones. IFN-γ production by Thl clones was detectable in almost all cells by 4 h, and it continued in most cells for >24 h. IL-4 production in Th2 cells peaked at 4 h, but declined rapidly. In Th0 cells containing both cytokines, fewer cells produced IFN-γ which did not appear until IL-4 synthesis declined. Cocultivation of clones showed no such cross-regulation. Antigen stimulation of transgenic T cells expressing an ovalbumin-specific T cell receptor generated Th2 cells, probably as a result of endogenous IL-4 production. Addition of IL-12 and/or anti-IL-4 caused Thl cells to develop, while some Th0 cells were seen when IL-12 alone was added. These results show that stimulation in the presence of polarizing stimuli results in cells producing either IFN- T or IL-4, but that coproduction can occur in rare cells under defined conditions.