IGF-I is mitogenic for the bovine mammary epithelial cell line MAC-T. In addition, IGF-I specifically upregulates IGFBP-3 synthesis in these cells. To investigate this effect on cell growth and IGF-I responsiveness, cell lines were developed that constitutively express IGFBP-3. MAC-T cells transfected with IGFBP-3 (ϩBP3) or vector alone (Mock) grew similarly over 7 days in 10 or 1% fetal calf serum. Basal DNA synthesis was lower (70%) in ϩBP3 cells compared to Mock cells. However, DNA synthesis was increased by IGF-I (1-50 ng/ml) relative to untreated controls to a greater extent in ϩBP3 cells compared to Mock cells. IGF-I (20 ng/ml) increased DNA synthesis 11-and threefold in ϩBP3 and Mock cells, respectively. Additionally, ϩBP3 cells were more sensitive to the lower concentrations of IGF-I (1-5 ng/ml). In contrast, preincubation of Mock cells with exogenous IGFBP-3 did not enhance responsiveness or sensitivity to IGF-I. Basal DNA synthesis was unaffected by either an IGF neutralizing antibody or exogenous IGFBP3, indicating the differences observed between ϩBP3 and Mock cells were not attributable to sequestration of endogenous IGF-I by IGFBP-3. There were no differences between ϩBP3 and Mock cells in IGF-I receptor number or affinity. DNA synthesis was also increased in ϩBP3 cells, compared to controls, in response to 5 g/ml insulin and 2.5 ng/ml Long R 3 IGF-I, indicating that the potentiated response did not require an interaction with IGFBP-3. These results suggest that IGF-I regulation of IGFBP-3 represents a regulatory loop, the function of which is to increase IGF-I bioactivity, using a mechanism that does require an IGF-I-IGFBP-3 interaction.