Several groups of proteolytic enzymes are able to degrade components of the extracellular matrix. During atherosclerosis, matrix remodeling is believed to influence the migration and proliferation of cells within the plaque. In the present study, gene expression of several proteases and their inhibitors was analyzed during the development of atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice. Quantitative real-time polymerase chain reaction was used to study gene expression of proteases after 10 and 20 weeks in ApoE−/− and C57BL/6 mice and in atherosclerotic lesions and nonaffected regions of the same ApoE−/− mouse. Some of the differentially expressed proteolytic enzymes were studied by immunohistochemistry. The matrix metalloproteinase (MMP)-9 and its inhibitor TIMP-1 were differentially expressed and the expression increased with time. Urokinase-type plasminogen activator showed no major expression. In contrast, cathepsins B, D, L, and S all showed strong and increased expression in ApoE−/− mice compared to C57BL/6 mice whereas the expression of their inhibitor, cystatin C, did not differ between the two mouse strains. The expression of cathepsins was mainly localized to the lesions and not to nonaffected regions of the aorta of ApoE−/− mice. Furthermore, cathepsin expression was similar to the expression of the macrophage marker macrosialin (CD68) although expression of cathepsins B, D, and L could be demonstrated in healthy C57BL/6 mice and in nonaffected vessel segments of atherosclerotic ApoE−/− mice. Cathepsin S mRNA expression was restricted to lesions of ApoE−/− mice. Furthermore, cathepsin S was the only cathepsin that was expressed in the media and absent in lipid-rich regions. All cathepsins studied showed intimal expression, the degree and localization of which differed between individual cathepsins. In conclusion, increased expression of several cathepsins in atherosclerotic lesions suggests that these proteases may participate in the remodeling of extracellular matrix associated with the atherosclerotic process.