Insulin-like growth factor binding protein-3 (IGFBP-3) can inhibit cell growth by directly interacting with cells, as well as by forming complexes with IGF-I and IGF-II that prevent their growth-promoting activity. The present study examines the mechanism of inhibition of DNA synthesis by IGFBP-3 in CCL64 mink lung epithelial cells. DNA synthesis was measured by the incorporation of 5-bromo-2'-deoxyuridine, using an immunocolorimetric assay. Recombinant human IGFBP-3 (rh[N109D,N172D]IGFBP-3) inhibited DNA synthesis in proliferating and quiescent CCL64 cells. Inhibition was abolished by co-incubation of IGFBP-3 with a 20% molar excess of Leu 60 -IGF-I, a biologically inactive IGF-I analogue that binds to IGFBP-3 but not to IGF-I receptors. DNA synthesis was not inhibited by incubation with a preformed 1:1 molar complex of Leu 60 -IGF-I and IGFBP-3, indicating that only free IGFBP-3 inhibits CCL64 DNA synthesis. Inhibition by IGFBP-3 is not due to the formation of biologically inactive complexes with free IGF, since endogenous IGFs could not be detected in CCL64 conditioned media; any IGFs that might have been present could only have existed in inactive complexes, since endogenous IGFBPs were present in excess; and biologically active IGFs were not displaced from endogenous IGFBP complexes by Leu 60 -IGF-I. After incubation with CCL64 cells, 125 I-IGFBP-3 was covalently cross-linked to a major โซ-004ูโฌkDa complex. This complex co-migrated with a complex formed after incubation with 125 I-labeled transforming growth factor-โค (TGF-โค) that has been designated the type V TGF-โค receptor. 125 I-IGFBP-3 binding to the โซ-004ูโฌkDa receptor was inhibited by co-incubation with unlabeled IGF-I or Leu 60 -IGF-I. The ability of Leu 60 -IGF-I to decrease both the inhibition of DNA synthesis by IGFBP-3 and IGFBP-3 binding to the โซ-004ูโฌkDa receptor is consistent with the hypothesis that the โซ-004ูโฌkDa IGFBP-3 receptor mediates the inhibition of CCL64 DNA synthesis by IGFBP-3.