Bone marrow endothelial cells are the essential component of the bone marrow microenvironment. They produce many kinds of cytokines, including stimulators and inhibitors. Many researchers have suggested that in the presence of endothelial cell layer, CD34+CD38- cells are capable of expansion. The ability of the endothelial cell layer to protect hematopoietic stem cells from extensive differentiation may be related to the inhibitors derived from endothelial cells. The aim of the present study was to determine whether the inhibitors thymosin beta4 and AcSDKP are elaborated by murine bone marrow endothelial cells. Murine bone marrow endothelial cells (mBMECs) were cultured in serum-free conditioned medium. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the differential expression of the thymosin-beta gene, and reverse phase high-performance chromatography (HPLC) and mass spectroscopy were used to determine the concentration of thymosin beta4 (Tbeta4) and AcSDKP in EC lysate and in the medium (mBMEC-CM). Colony-forming unit granulocyte-macrophage (CFU-GM) colony assays were used to examine the effect of components (mw 3-10 kD, <3 kD) of mBMEC-CM, thymosin beta4, and AcSDKP on the proliferation of hematopoietic cells.mBMECs expressed Tbeta4 mRNA. In EC lysate and mBMEC-CM, Tbeta4 and AcSDKP were detected. After adding protease inhibitors, the concentration of Tbeta4 in EC lysate increased significantly, while the concentration of AcSDKP decreased. mBMEC-CM (mw 3-10 kD) had no effect on the formation of CFU-GM. However, mBMEC-CM (mw <3 kD) could inhibit the growth of CFU-GM. Tbeta4 (10(-11) approximately 10(-7)mol/L) and AcSDKP (10(-11) approximately 10(-5)mol/L) had dose-dependent inhibitory effects on the growth of CFU-GM. Angiotensin converting enzyme (ACE), the enzyme degrading AcSDKP, could partially eliminate the inhibitory effect of mBMEC-CM (mw <3 kD) on CFU-GM.BMECs express and secrete Tbeta4 and AcSDKP. Tbeta4 exists in the 3-10 kD component of mBMEC-CM, while AcSDKP exists in the <3 kD component of ECCM. Both components exert inhibitory effects on the proliferation of hematopoietic progenitors.