Proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3), the major carrier of IGFs in the circulation, is an essential mechanism to regulate IGF bioavailability. To analyze naturally occurring IGFBP-3 fragments a peptide library established from human hemofiltrate was screened. Three IGFBP-3 fragments were detected with apparent molecular masses of 34, 16, and 11 kDa. Mass spectrometric and sequence analysis identified the 16 and 11 kDa peptides as glycosylated and non-glycosylated N-terminal fragments spanning residues Gly1-Ala98 of IGFBP-3. Both the circulating forms and those secreted from IGFBP-3(1-98) overexpressing cells bound IGF. Additionally, two smaller fragments (IGFBP-3(139-157) and IGFBP-3(139-159)) were identified in the hemofiltrate. The data indicate that proteolysis of circulating IGFBP-3 occurs in the variable domain at residues alanine 98, phenylalanine 138, glutamine 157, and tyrosine 159.