This study investigates the inhibition of endothelial cell (EC) movement by pericytes and smooth muscle cells (SMCs) in co-culture. When a confluent monolayer of bovine aortic endothelial (BAE) cells is wounded, ECs move into the denuded area. However, when pericytes or SMCs are plated into the wound area at low density, they block EC movement. This inhibition is dependent on the number of plated cells and their contact with ECs. Transforming growth factor-β1 (TGF-β1), a known inhibitor of EC movement, is activated in BAE cell-pericyte or BAE cell-SMC co-cultures, and this activation is mediated by plasmin. The activation of latent TGF-β1-like molecules produced by BAE cells, pericytes, and SMCs is crucial for the inhibition of EC movement. The results suggest that the interaction between ECs and pericytes or SMCs involves the activation of latent TGF-β1, which is regulated by plasmin, and that this process may have implications for the regulation of EC movement and proliferation in various physiological and pathological conditions.This study investigates the inhibition of endothelial cell (EC) movement by pericytes and smooth muscle cells (SMCs) in co-culture. When a confluent monolayer of bovine aortic endothelial (BAE) cells is wounded, ECs move into the denuded area. However, when pericytes or SMCs are plated into the wound area at low density, they block EC movement. This inhibition is dependent on the number of plated cells and their contact with ECs. Transforming growth factor-β1 (TGF-β1), a known inhibitor of EC movement, is activated in BAE cell-pericyte or BAE cell-SMC co-cultures, and this activation is mediated by plasmin. The activation of latent TGF-β1-like molecules produced by BAE cells, pericytes, and SMCs is crucial for the inhibition of EC movement. The results suggest that the interaction between ECs and pericytes or SMCs involves the activation of latent TGF-β1, which is regulated by plasmin, and that this process may have implications for the regulation of EC movement and proliferation in various physiological and pathological conditions.