We have examined the biologic and physical properties of a human T-lymphocyte granulocyte-macrophage colony-stimulating factor (CSF). The source of the factor is a T-lymphoblast cell line (Mo) that was derived from a patient with a T-cell variant of hairy-cell leukemia. The Mo line constitutively produces a number of lymphokines that are normally produced by mitogen-stimulated T lymphocytes. Medium conditioned by Mo cells grown in the absence of serum is especially rich in CSF activity, and using this source we have purified the CSF to a specific activity of about 3.5 x 10(6) colonies per 10(5) Ficoll-Hypaque-separated human bone marrow cells plated per mg protein. The Mo CSF stimulates the formation of both granulocyte and macrophage colonies in vitro (in about equal numbers) and it has a relatively steep dose-response curve. Both the crude and purified preparations stimulated the formation of eosinophil as well as neutrophil colonies; it is unclear whether this is due to the presence of multiple factors with similar physical properties or a single factor with multiple activities. The CSF has little stimulating activity for mouse bone marrow progenitors. Physically, the Mo CSF is an acidic glycoprotein of molecular weight about 34,000. It binds to concanavalin A-Sepharose, is unusually resistant to denaturing agents and heat treatment, and is not inactivated in the presence of sulfhydryl reagents. The Mo CSF is distinct from factors stimulating erythroid colony formation and inhibiting neutrophil migration that are also produced by Mo cells. It differs in several physical and biologic properties from other human CSFs that have been characterized.