The proapoptotic proteinase inhibitor TIMP-3 is the only molecule of this family thought to influence cell death. We examined epithelial apoptosis in TIMP-3-deficient mice during mammary gland involution. Lactation was not affected by the absence of TIMP-3, but glandular function, as measured by gland-to-body weight ratio and production of beta-casein, was suppressed earlier during post-lactational involution than in controls. Histological examination revealed accelerated lumen collapse, alveolar-epithelial loss, and adipose reconstitution in Timp-3(-/-) females. Epithelial apoptosis peaked on the first day of involution in Timp-3-null glands but at day 3 in wild-type littermates. Unscheduled activation of gelatinase-A was evident by zymography and correlated with earlier fragmentation of fibronectin in Timp-3(-/-) mammary. To obtain independent evidence of the proapoptotic effects of TIMP-3 deficiency, we introduced recombinant TIMP-3-releasing pellets into regressing Timp-3(-/-) mammary tissue and showed that this treatment rescued lumen collapse and epithelial apoptosis. Ex vivo, involuting Timp-3(-/-) mammary tissue demonstrated accelerated epithelial apoptosis that could be reduced by metalloproteinase inhibition. The physiological relevance of TIMP-3 became apparent as Timp-3(-/-) dams failed to reestablish lactation after brief cessation of suckling. Thus, TIMP-3 is a critical epithelial survival factor during mammary gland involution.