We examined the expression level of several genes that regulate different steps in metastasis formation in formalin-fixed, paraffin-embedded biopsies of 189 primary human gastric carcinomas prior to surgical resection in patients in whom lymph node metastasis was not evident by endoscopic ultrasound or computed tomography (CT) scan. The expressions of epidermal growth factor receptor (EGF-R), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and E-cadherin were examined by a colorimetric in situ mRNA hybridization technique. The integrity of the mRNAs was verified, leaving 161 (85.2%) patients for study. After gastrectomy, 82 patients had positive lymph nodes and 79 patients had negative lymph nodes. The concurrent expression levels of MMP-2 and E-cadherin mRNAs were significantly higher and lower, respectively, in the metastatic tumors than the non-metastatic tumors. Expression of EGF-R and VEGF was not different between the metastatic and non-metastatic tumors. However, when only the intestinal-type of gastric cancer was evaluated, the level of VEGF mRNA was significantly higher in tumors associated with lymph node metastasis than in those without metastasis. However, a high MMP-2:E-cadherin ratio significantly correlated with lymph node metastasis in both types of gastric cancer. These results suggest that multiparametric in situ hybridization analysis for several metastasis-related genes may allow the preoperative prediction of lymph node metastasis from gastric cancer.