IGF-binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells directly without sequestering IGF-I and IGF-II. The molecular mechanisms responsible for the IGF-independent actions of IGFBP-3 remain unclear. IGFBP-3, a secreted protein, can be internalized and translocate to the nucleus. It binds to the nuclear retinoid receptor RXR-. Binding to RXR- has been proposed to be required for IGFBP-3 to induce apoptosis. The present study tests this hypothesis in PC-3 human prostate cancer cells. PC-3 cells express RXR-, and apoptosis is induced by incubation with RXR-specific ligand. A COOH-terminal region in IGFBP-3 (residues 215–232) contains a nuclear localization signal (NLS) and binding domains for RXR- and heparin (HBD). Different combinations of the 11 amino acids in this region that differ from IGFBP-1, a related IGFBP which does not localize to the nucleus or bind RXR-, were mutated to the IGFBP-1 sequence. By confocal imaging, mutation of residues 228-KGRKR-232 in nonsecreted IGFBP-3 diminished its nuclear localization. IGFBP-3 binding to GST-RXR- only was lost when all 11 sites were mutated (HBD-11m-IGFBP-3). Expressed nuclear RXR- did not transport cytoplasmic IGFBP-3 NLS mutants that can bind RXR- to the nucleus even after treatment with RXR-ligand. Expressed HBD-11m-IGFBP-3 still induced apoptosis in PC-3 cells in an IGF-independent manner as determined by flow cytometric analysis of Annexin V staining. We conclude that in PC-3 cells RXR- is not required for the nuclear translocation of IGFBP-3, and that IGFBP-3 can induce apoptosis in human prostate cancer cells without binding RXR-.