To assess the participation of the plasminogen activation system in the invasiveness of esophageal squamous cell carcinoma, we performed immunohistochemistry and in situ hybridization to study the distribution of a urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR), and plasminogen activator inhibitor-2 (PAI-2). u-PA and PAI-2 were expressed heterogeneously in cancer cells, and restricted expression was found in stromal cells, especially fibroblasts, that were located in the immediate proximity of the cancerous cells. u-PAR was found only in cancer cells located at the periphery of tumors. Compared with patients with u-PA-negative cancer cells, patients with u-PA-positive cancer cells more frequently showed a neoplastic invasion beyond the muscularis propria and lymph node metastases. They also showed a significantly shorter 5-year overall survival. Patients with PAI-2-positive fibroblasts showed significantly lower levels of local invasiveness, represented by a neoplastic invasion beyond the muscularis propria, than those who were PAI-2 negative. Our results suggest that the expression of u-PA in esophageal squamous cell carcinoma is predictive of poor survival, whereas the expression of PAI-2 in the fibroblasts surrounding them is protective. An analysis of u-PA and PAI-2 expression in cancer cells and their surrounding fibroblasts may be useful for predicting the prognosis of patients with esophageal squamous cell carcinoma.