Collagen I regulates matrix metalloproteinase-2 activation in osteosarcoma cells independent of S100A4. This work investigates the effect of cell-collagen I interactions on the synthesis and activation of MMP-2, as well as synthesis of MT1-MMP and TIMP-1, by using an in vitro model with 3D fibrillar and 2D monomeric collagen. In order to reveal whether the metastasis-associated protein S100A4 can influence the cell's response to the two forms of collagen, osteosarcoma cell lines with high and low endogenous levels of S100A4 were used. Attachment of osteosarcoma cells to 3D fibrillar and 2D monomeric collagen resulted in opposite effects on MMP-2 activation. Attachment to 3D fibrillar collagen decreased activation of proMMP-2, with a corresponding reduction in MT1-MMP. By contrast, attachment to monomeric collagen increased the amount of fully active MMP-2. This was caused by a reduction in TIMP-1 levels when cells were attached to monomeric 2D collagen. The effect of collagen on proMMP-2 activation was independent of endogenous S100A4 levels, whereas synthesis of TIMP-1 was dependent on S100A4. When cells were attached to monomeric collagen, cells with a high level of S100A4 showed a greater reduction in the synthesis of TIMP-1 than did those with a low level of S100A4. Taken together, this study shows that synthesis and activation of MMP-2 is affected by interactions between osteosarcoma cells and collagen I in both fibrillar and monomeric form.