The insulin-like growth factor (IGF)-binding proteins (IGFBPs) carry IGFs in serum and regulate their activity and bioavailability. The main IGFBP in serum, IGFBP-3, is known to form a 150-kDa complex with IGFs and the acid-labile subunit (ALS). We investigated the binding of IGFBP-3 to additional association proteins in human serum (IGFBP-3 APs). Ligand blots, column chromatography, and affinity cross-linking experiments revealed the specific binding of IGFBP-3 to at least three novel serum proteins. These techniques demonstrated the presence of proteins with molecular masses of 70, 100, and 150 kDa that bind IGFBP-3 with high affinity. Serum ALS migrated separately (at 88 kDa) from the novel IGFBP-3 APs (as evident by Western immunoblot), and bound IGFBP-3 weakly (by reverse ligand blots). We also demonstrated that large amounts of one of the IGFBP-3 APs and small amounts of ALS were coimmunoprecipitated with IGFBP-3 from human serum. Similar to ALS, these IGFBP-3 APs are acid labile and lose their IGFBP-3 binding capacity after exposure to low pH. We conclude that there are several serum proteins in addition to ALS and IGFs that bind IGFBP-3 with high affinity. These IGFBP-3 APs may serve as an additional reservoir of IGFBP-3 or modulate its functions.