To analyze the molecular mechanisms of coronary vessel formation, we performed in vitro experiments on explant cultures of proepicardial organs (PEOs) excised from embryos taken from 9.5-day pregnant mice. When plated on coverglasses coated with rat tail collagen I, fibronectin, or laminin, PEO cells spread and formed an epithelial sheet. When PEOs were cultured on collagen gel in the presence of fetal calf serum (FCS), small projections were seen around the explants 3 days after plating. Around day 6, cord-like structures began to grow from the explants, gradually elongating, increasing in number, and forming a branching network. Histological sections demonstrated that the cells migrated into the gel and formed tube-like structures similar to the vascular channels of the embryonic heart. The cells lining the lumen of the tube-like structures were positive for platelet endothelial cell adhesion molecule (PECAM). Reverse transcriptase-polymerase chain reaction analyses demonstrated that the expression of PECAM, basic fibroblast growth factor (bFGF), and smooth muscle 22-alpha (SM22alpha) was upregulated in association with the tube formation, whereas the expression of Flk-1, Flt-1, and hepatocyte growth factor (HGF) was gradually downregulated. Vascular endothelial growth factor (VEGF) was continuously expressed during the culture. These changes were not observed when PEOs were explanted without FCS. Furthermore, addition of any one or combinational addition of the growth factors, including bFGF, VEGF, or HGF, did not induce tube formation. These results suggest that PEOs contain precursor cells of coronary vasculature and that vasculogenesis may be simultaneously regulated by multiple factors.