Objective: While invading and transiting the dermis melanoma cells interact with the extracellular matrix (ECM) and fibroblasts. To study the roles ECM components, such as decorin and chondroitin sulfates, and fibroblasts play in melanoma cell migration and invasion we established a co-culture model system that contained murine fibroblasts, their collagen-rich matrix and mouse melanoma cells (B16V). Methods: Mouse fibroblasts were cultured in the presence of ascorbate-2-phosphate for 14 days. B16 melanoma cells were then seeded on the matrix created by the fibroblasts. Matrix components were detected by immunofluorescence. B16 cell migration and invasion were monitored by time lapse video-microscopy. Cytosolic pH was measured ratiometrically using the proton-sensitive, fluorescent dye BCECF. Westen blots and zymography assays were performed in order to determine the release and activity of matrix metallo-proteases (MMP-2) in response to the crosstalk between B16 cells and fibroblasts. Results: Major components of the fibroblast ECM were collagen I, fibronectin, fibromodulin, and lumican. In semi-thin sections, invasive B16 cells were detected inside the ECM. Migration speed and the distance traveled were significantly higher on the living matrix than on artificial, reconstituted matrices. Both migration and invasion decreased by more than 50% when B16 cells were exposed to either the Na⁺/H⁺ exchanger (NHE1) inhibitor cariporide or to the added solute ECM components decorin or chondroitin sulfates. Decorin and cariporide led to a reversible, chondroitin-6-sulfate to a non-reversible acidification of the cytosol. Cariporide, decorin and chondroitin-6-sulfate hindered the conversion of pro-MMP-2 to the mature MMP2. Conclusion: The living matrix is an appropriate tool to analyze migration, invasion and MMP release depending on (i) cell matrix interactions and (ii) the crosstalk between the invasive cells and those surrounded by their self-made matrix.