ADAM-9 belongs to a family of transmembrane disintegrin-containing metalloproteinases (ADAMs) involved in protein ectodomain shedding and cell-cell and cell-matrix interactions. However, the specific biological functions of ADAM-9 are still unclear. The aim of this study was to analyze the expression of ADAM-9 in melanoma in vivo and in melanoma cell lines in vitro. In melanoma ADAM-9 protein expression appeared to be restricted to the melanoma cells within the invading front. Interestingly, ADAM-9 protein was detected in the melanoma cells and in peritumoral stromal fibroblasts, while it was absent in fibroblasts distal to the tumor site. RNA analysis of melanoma cell lines with different invasive abilities showed ADAM-9 expression in varying amounts in all cell lines, independent of their invasive and metastatic capacities. In MV3 melanoma cells, ADAM-9 expression did not depend on homotypic cell-cell contact and on cell-matrix interaction when the cells were cultured on planar extracellular matrix components. However, we observed downregulation of ADAM-9 mRNA expression upon culture of melanoma cells within 3-dimensional lattices composed of fibrillar type I collagen, whereas culture within gels consisting of the polysaccharide alginate did not alter transcript levels. These results identified fibrillar collagen type I as a key factor in ADAM-9 regulation by cell-matrix interactions. Interestingly, we also observed a 3-fold downregulation of ADAM-9 transcript levels upon treatment with interleukin (IL)-1alpha, a proinflammatory cytokine known to induce expression of other ADAM and matrix metalloproteinase (MMP) family members. In summary, our data suggest a novel role of fibrillar collagen and of soluble factors for the regulation of ADAM-9 expression in vitro.