Human insulin-like growth factor II (IGF-II) has been purified to homogeneity from bone which contained 10–15 times more IGF-II than insulin-like growth factor-I (IGF-I). After extraction of IGF-II by demineralization of human bone powder with 10% EDTA containing 4 M guanidine HCl at pH 7.4, IGF-II was separated from IGF binding proteins by hydroxylapatite chromatography in the presence of 4 M guanidine HCl. The hydroxylapatite unbound fraction containing IGF-II was purified by affinity chromatography using Sm 1.2. monoclonal antibodies, which bind both IGF-I and IGF-II. The final purification of IGF-II was achieved by FPLC mono S ion-exchange chromatography in which IGF-II was separated from IGF-I. Human IGF-II thus purified was shown to be pure by (1) HPLC reverse-phase chromatography, (2) SDS-PAGE, and (3) N-terminal amino acid sequence.From 300 g of bone, 0.18 mg IGF-II was obtained with an overall recovery of 42%. These studies demonstrate the usefulness of (1) bone as a source for IGF-II purification and (2) antibodies that cross-react with both IGF-I and IGF-II for affinity purification of IGFs.