Th2 cells can be subdivided into subpopulations depending on the level of a cytokine and the subsets of cytokines they produce. We have recently identified the ETS family transcription factor PU. 1 as regulating heterogeneity in Th2 populations. To define additional factors that might contribute to Th2 heterogeneity, we examined the PU. 1 interacting protein IFN-regulatory factor (IRF) 4. When Th2 cells are separated based on levels of IL-10 secretion, IRF4 expression segregates into the subset of Th2 cells expressing high levels of IL-10. Infection of total Th2 cells, and IL-10 nonsecreting cells, with retrovirus-expressing IRF4, resulted in increased IL-4 and IL-10 expression, no change in IL-5 or IL-13 production and decreased Il9 transcription. Transfection of an IRF4-specific small interfering RNA into Th2 cells decreases IL-10 production. IRF4 directly binds the Il10 gene as evidenced by chromatin immunoprecipitation assay, and regulates Il10 control elements in a reporter assay. IRF4 interacts with PU. 1, and in PU. 1-deficient T cells there was an increase in IRF4 binding to the Il10 gene, and in the ability of IRF4 to induce IL-10 production compared with wild-type cells and Il10 promoter activity in a reporter assay. Further heterogeneity of IRF4 expression was observed in Th2 cells analyzed for expression of multiple Th2 cytokines. Thus, IRF4 promotes the expression of a subset of Th2 cytokines and contributes to Th2 heterogeneity.