We have demonstrated a 50-fold increase in the concentration of insulin-like growth factor-binding protein-5 (IGFBP-5) in milk after 2 days of mammary involution induced by removal of the suckling young. IGFBP-5 was identified by its immunoreactivity with an antiserum to IGFBP-5 and was shown by in situ hybridization to be synthesized by the secretory epithelial cells undergoing apoptosis. Smaller increases in IGFBP-2 and -4 messenger RNAs (mRNAs) were also evident, but neither protein could be detected on Western ligand blots of milk. Preliminary evidence failed to detect mRNAs for IGFBP-1, -3, or -6. The large increase in IGFBP-5 concentrations in milk from involuting mammary glands was inhibited by 90% if the dams received concurrent PRL injections for 2 days, but was unaffected by GH, progesterone, corticosterone, or an antiserum to insulin-like growth factor I (IGF-I). In lactating rats allowed to continue nursing their young, 17beta-estradiol failed to affect IGFBP-5 concentrations, whereas in animals that had half the teats sealed to prevent milk removal, IGFBP-5 concentrations increased 5- to 10-fold in the sealed gland compared with those in the contralateral gland where milk removal continued. The changes in IGFBP-5 concentrations in milk were accompanied by similar changes in steady state mRNA levels of IGFBP-5 in mammary tissue. We have previously shown that PRL inhibits apoptosis and involution of the mammary gland, whereas teat sealing has the opposite effect. We, therefore, propose that IGFBP-5 serves to inhibit IGF-I-mediated cell survival, but that it is normally suppressed by PRL and milk removal. Although IGFBP-5, when bound to extracellular matrix, augments the action of IGF, we believe that in the involuting mammary gland IGFBP-5 inhibits IGF action by interacting with casein micelles, which contain calcium phosphate nanoclusters, thereby preventing IGF interaction with IGF receptors. This is analogous to the interaction of IGFBP-5 with hydroxyapatite, which serves to sequester IGFs in bone. IGFBP-5 may, in fact, play a central role in inducing apoptosis, as it is also up-regulated in involuting prostate and thyroid glands as well as in atretic ovarian follicles.