We used a customized Affymetrix protease microarray (Hu/Mu ProtIn chip) designed to distinguish human and mouse genes to analyze the expression of proteases and protease inhibitors in lung cancer. Using an orthotopic lung cancer model, we showed that murine matrix metalloproteinase (MMP)-12, MMP-13, and cathepsin K were up-regulated in tumor tissue compared with normal mouse lung. To determine the relevance of stromal proteases detected using this model system, we compared the results to an analysis of human lung adenocarcinoma specimens using the U133 Plus 2.0 Affymetrix microarray. MMP-12, MMP-13, and cathepsin K showed an increase in expression in human tumors compared with normal lung similar to that seen in the orthotopic model. Immunohistochemical analysis confirmed MMP-12 expression in the stroma of human lung tumor samples. To determine the biological relevance of stromal MMP-12, murine Lewis lung carcinoma cells were injected into the tail vein of syngeneic wild-type (WT) and MMP-12-null mice. MMP-12-null and WT mice developed equivalent numbers of lung tumors; however, there was a 2-fold increase in the number of tumors that reached >2 mm in diameter in MMP-12-null mice compared with WT controls. The increase in tumor size correlated with an increase in CD31-positive blood vessels and a decrease in circulating levels of the K1-K4 species of angiostatin. These results show a protective role for stromal MMP-12 in lung tumor growth. The use of the Hu/Mu ProtIn chip allows us to distinguish tumor- and host-derived proteases and guides the further analysis of the significance of these genes in tumor progression.