AbstractMM is a B‐cell malignancy mainly characterized by monoclonal expansion of plasma cells in the BM, presence of paraprotein in serum and occurrence of osteolytic bone lesions. MMPs are a family of proteolytic enzymes that can contribute to cancer growth, invasion, angiogenesis, bone degradation and other processes important in the pathogenesis of MM. We investigated MMP‐9 production in the 5T33MM murine model. Expression of MMP‐9 protein in supernatant and cell extracts was analyzed by gelatin zymography. The in vitro, stroma‐independent variant 5T33MMvt showed no protein expression of MMP‐9 in contrast to in vivo growing MM cells, 5T33MMvv. However, when 5T33MMvt cells were injected into naive mice and isolated after tumor take (5T33MMvt‐vv), they secreted a significant amount of MMP‐9. These results were confirmed by specific staining of cytospins with an anti‐MMP‐9 antibody. The MMP‐9 production by 5T33MMvt‐vv cells disappeared when the cells were recultured in vitro. These data demonstrated that upregulation of MMP‐9 occurs in vivo and that this process is dependent on the microenvironment. Cocultures of 5T33MMvt cells with STR10 BMECs induced MMP‐9 in MM cells, as determined by both gelatin zymography and flow‐cytometric analysis. In conclusion, our results demonstrate that MMP‐9 production by MM cells is upregulated in vivo by the interaction of MM cells with BMECs. © 2002 Wiley‐Liss, Inc.