Expression of adhesion receptor integrin alphavbeta3 in an activated functional form strongly promotes metastasis in human breast cancer cells. Here, we report that alphavbeta3 cooperates with matrix metalloproteinase type 9 (MMP-9) in breast cancer cell migration. This cooperation is regulated by the activation state of the integrin. Expression of activated alphavbeta3 in metastatic variants of MDA-MB 435 human breast cancer cells and primary metastatic cells from breast cancer patients strongly enhanced migration toward vitronectin and fibrinogen. This enhancement was mediated by a soluble factor produced by breast cancer cells expressing activated alphavbeta3. When transferred, this factor also up-regulated alphavbeta3-dependent migration of breast cancer cells that express the nonactivated integrin. The factor was identified as metalloproteinase MMP-9. Whereas all tested breast cancer cell variants produced latent MMP-9, only those with activated alphavbeta3 produced the mature form of this metalloproteinase. Recombinant mature MMP-9, but not latent MMP-9 or either form of MMP-2, enhanced alphavbeta3-dependent breast cancer cell migration. The migratory response was inhibited by tissue inhibitors of metalloproteinase or when MMP-9 was depleted from the inducing supernatants. The results indicate a causal relationship between the expression of activated integrin alphavbeta3 and production of enzymatically active MMP-9 in metastatic breast cancer cells. These molecules cooperate to enhance breast cancer cell migration toward specific matrix proteins, and this may contribute to the strongly enhanced metastatic capacity of breast cancer cells that express activated alphavbeta3.