Insulin-like growth factor binding protein-3 (IGFBP-3) has antiproliferative effects in many cell types but paradoxical growth stimulation has also been reported. In early passages following transfection of T47D breast cancer cells with IGFBP-3 cDNA, the proliferation rate and serum-stimulated DNA synthesis were significantly reduced compared to control cells. Cell cycle analysis indicated that growth-inhibited IGFBP-3-producing cells accumulated in G1 phase. After several passages, the transfected cells became resistant to the inhibitory effects of IGFBP-3 and showed transiently enhanced proliferation rates despite an increased IGFBP-3 concentration in the medium. IGFBP-3 proteolysis did not account for its decreased antiproliferative activity in resistant cells. We hypothesize that development of resistance to the antiproliferative action of IGFBP-3 might be an important step in the malignant progression of breast cancer cells.