The tissue inhibitors of metalloproteinases 1–4 (TIMPs) have discrete regulatory roles in the activation of matrix metalloproteinase (MMP)-2 (gelatinase A), an important basement membrane-degrading MMP pivotal to tumor metastasis and angiogenesis. TIMP-2 binds to both the hemopexin C domain of progelatinase A and the active site of membrane type-1 (MT1) MMP. This trimeric complex presents the cell surface-bound gelatinase A zymogen to a free MT1-MMP molecule for activation. To investigate the role of TIMP-4 in the activation process, we developed a new procedure for the expression and purification of recombinant human TIMP-4 from baby hamster kidney cells. The recombinant TIMP-4 was a potent inhibitor of gelatinase A {apparent K_i [K_i(app.)] ≤ 9 pm; k_on (association rate constant), 4.57 ± 0.13 × 10⁶ m⁻¹s⁻¹} and was less dependent upon hemopexin C domain interactions than TIMP-2 in its mode of binding and inhibition. Unlike TIMP-1, TIMP-4 strongly inhibited MT1-MMP (K_i(app.) ≤ 100 pm; k_on, 3.49 ± 0.34 × 10⁶ m⁻¹s⁻¹) and blocked the concanavalin A-induced cellular activation of progelatinase A. In concanavalin A-stimulated homozygous Timp2 −/− fibroblasts or unstimulated MT1-MMP-transfected Timp2 −/− cells, which cannot activate progelatinase A, activation was restored by the addition of 0.3–5 nm TIMP-2 but not by TIMP-4, unequivocally showing the TIMP-2 dependency of MT1-MMP-induced activation of gelatinase A and the fact that TIMP-4 cannot support activation. The dominance of TIMP-2 in the activation process was further supported by the preferential binding of TIMP-2 compared with TIMP-4 to the hemopexin C domain of progelatinase A in inhibitor mixtures and by the ability of TIMP-2 to displace TIMP-4 from the hemopexin C domain. Hence, TIMP-4 regulates gelatinase A activity by efficient inhibition of MT1-MMP-mediated activation and by inhibiting the activated enzyme and, thus, is a tumor resistance factor in the peritumor stroma.