Spleen cells from mice undergoing a parasite-induced eosinophilia were fused with an azaguanine-resistant subline of the thymoma BW5147. A stable T hybrid (NIMP-TH1) was isolated and selected by recloning repeatedly by limiting dilution. The hybrid nature of NIMP-TH1 was confirmed by its expression of both parental alleles of Thy-1 and by chromosome analysis (modal chromosome number 102). On stimulation with phorbol myristate acetate, this hybrid releases a soluble activity which acts as a stimulator of eosinophil differentiation in vitro. Addition of hybrid conditioned medium to bone marrow cultures results in a selective stimulation of eosinophil production with no detectable increase in neutrophil or macrophage differentiation. The lymphokines interleukin-2 (IL-2) and interferon (IFN) are undetectable in NIMP-TH1 conditioned media. Although at high concentrations NIMP-TH1 supernatants are able to support very low levels of DNA synthesis in an IL-3-dependent cell line, and IL-3 appears to support low levels of eosinophil differentiation, dose-response curves show that the factor produced by NIMP-TH1 can be clearly segregated from IL-3 by its marked specificity for cells belonging to the eosinophil lineage. The factor present in these supernatants has been provisionally termed eosinophil differentiation factor (EDF).