We derived a permanent human T lymphocyte cell line that elaborates a potent colony-stimulating activity (CSA). The line was established with spleen cells from a patient with a T lymphocyte variant of hairy-cell leukemia. These cells form rosettes with sheep erythrocytes, show a proliferative response to phytohemagglutinin, and are lysed by antithymocyte globulin. They do not synthesize immunoglobulin, nor do they contain Epstein-Barr virus. CSA is regularly detected in the supernatant medium after 3 days culture. In the presence of PHA there is augmented elaboration of CSA; maximal activity is reached by 2 days and is 20% greater than that produced by a feeder layer of 1 X 10(6) peripheral blood leukocytes. One microliter of the supernatant material stimulated colony formation from the light-density nonadherent fraction of human bone marrow; there was maximal activity between 10 and 50 microliter/ml. Conditioned medium from these cells has little effect in stimulating CFU-C from murine bone marrow. The availability of a human T lymphocyte line producing CSA will provide a source for large quantities of the lymphocyte-derived hormone and permit a definition of factors modulating the interaction of T lymphocytes with granulocyte and monocyte stem cells.