Endothelial progenitor cells (EPCs) from the bone marrow play an important role in vascular response to injury and ischemia. The mediators involved in the mobilization, recruitment, proliferation and differentiation of EPCs are not fully understood. In this study, the role of coagulation factor thrombin and protease-activated receptor-1 (PAR-1) on bone marrow-derived cell proliferation and differentiation was investigated. Bone marrow cells (BMCs) were isolated from C57/BL6 mice and plated on fibronectin-coated flasks. Cell characteristics, proliferation and the expression of endothelial cell markers were determined using immunohistochemistry, thymidine uptake and fluorescence activated-cell sorting (FACS), respectively. The results show that thrombin stimulated enrichment of bone marrow cells with endothelial morphology, exhibiting acetylated-low-density lipoprotein (LDL) uptake and isolectin staining. Thrombin or PAR-1-activating peptide produced a 2- to 3-fold increase in the total number of cells as well as an increase in vascular endothelial (VE)-cadherin-positive cells. Thrombin treatment of VE-cadherin-negative cells prepared after cell sorting resulted in the generation of 3- to 4-fold higher VE-cadherin-positive cells than the untreated cultures. Increase in VE-cadherin-positive cells was inhibited by hirudin and efegatran. These results provide first evidence for a novel activity of thrombin and PAR-1 on bone marrow progenitor cell proliferation and EPC differentiation, and suggest their potential role in vascular regeneration and recanalization of thrombus.