In order to identify regions in insulin-like growth factor binding protein-1 involved in the binding of IGFs, we tested three monoclonal antibodies, designated MAb A, B, and C on their interference with IGF-binding. Monoclonal A interfered with the binding of IGF to IGFBP-1 as determined by immunoprecipitation whereas monoclonal B and C did not. Furthermore MAb A was found to abolish IGFBP-1 inhibition of IGF stimulation in an in vitro proliferation assay. The epitopes of all three monoclonal antibodies were found to be located within the C-terminal part of IGFBP-1. The regions surrounding residue 188-196 and 222-227 are especially important for antibody recognition. These results indicate that MAb A functionally interferes with the binding of IGF to IGFBP-1. Furthermore, we suggest that part of the epitope of MAb A is located at or sterically near the IGF binding domain of IGFBP-1.