Latent transforming growth factors beta (TGF-beta) are easily detectable in embryonic and adult hematopoietic tissues and in vitro studies show that they are potent antagonists of lymphopoiesis and myelopoiesis when converted to biologically active form. To learn more about possible roles in hematopoiesis, active TGF-beta 1 was added to cultures prepared to support myeloid cells (Dexter conditions) or B lineage lymphocytes (Whitlock-Witte conditions) and studied in detail. Hematopoiesis was permanently arrested in Dexter cultures treated with 40 pmol/L (1 ng/mL) of active TGF-beta from initiation. In addition, adipogenesis was inhibited in a dose-dependent manner, and adherent layers from treated cultures were defective when recharged with fresh bone marrow cells. Ongoing neutrophil production was terminated in established cultures when addition of the factor was delayed for 8 weeks. In contrast, in experiments with Whitlock-Witte cultures, some of the flasks produced lymphocytes in the continuous presence of TGF-beta 1 (40 pmol/L). Lymphopoiesis was completely arrested by ten-fold higher concentrations, and this was most effective when added at the beginning of culture. Precursors of lymphocytes as well as the microenvironmental elements necessary for supporting their growth survived 2 weeks of cytokine treatment (400 pmol/L) in Dexter cultures. Normal outgrowth of lymphocytes occurred when the cultures were switched to Whitlock-Witte conditions. Surface marker expression on lymphocytes growing in TGF-beta resistant or previously treated cultures was not unusual. These studies demonstrate that TGF-beta is a negative regulator of hematopoiesis in long-term cultures and show that this includes effects on microenvironmental elements. At low concentrations, production of myeloid cells was preferentially affected.