Insulin-like growth factors (IGFs) circulate in human adult serum predominantly as a high mol wt complex of 150 kilodaltons (kDa), which is made up of three subunits: alpha, the acid-labile subunit, a glycoprotein of around 85-100 kDa; beta, the acid-stable IGF-binding protein subunit, which is the 40-kDa GH-dependent glycoprotein IGF-binding protein-3 (IGFBP-3); and gamma, the 7.5 IGF-I or -II peptide subunit. During human pregnancy, all three subunits are elevated when measured by RIA. However, recent ligand blotting studies have shown that IGFBP-3 is markedly reduced during pregnancy. When pregnancy serum is acidified and neutralized to inactivate endogenous alpha-subunit, ternary complex formation is normal with exogenous radiolabeled alpha-subunit, indicating functional IGFBP-3. We have attempted to clarify the status of IGFBP-3 and the high mol wt (alpha beta gamma) complex in human term pregnancy serum by further analyses. Term pregnancy (TP) or nonpregnancy (NP) pooled sera were fractionated on a S-300 neutral column. The high mol wt (125-150 kDa) and the low mol wt (30-40 kDa) IGF-IGFBP complexes were identified by both RIA for IGFBP-3 and ligand blotting; each was pooled separately. Half of each pool was lyophilized and rechromatographed in acid to separate the IGF-I peptides from their IGFBPs. The IGFBP fractions were recovered for further studies. When the IGFBPs from the high mol wt complex were cross-linked to [125I]IGF-I or -II, bands of 48, 34, 26, and 21 kDa, which were more intense with [125I]IGF-II, were observed, and all were immunoprecipitable by anti-IGFBP-3 antibody. The smaller forms were elevated in TP serum. Affinity cross-linking analysis with [125I]alpha-subunit showed that the IGFBPs from the high mol wt, but not the low mol wt, complex can reform the ternary 150-kDa complex when cold IGF-I or IGF-II is added exogenously. A smaller 130-kDa complex was also present, and it was the predominant form in TP serum. Both bands were immunoprecipitable by anti-IGFBP-3 antibody. When purified alpha-subunit or fractions from neutral Sephacryl S-300 chromatography were cross-linked to covalent [125I]IGF-II:IGFBP-3, a specific band at 150 kDa was observed with pure alpha-subunit as well as with S-300 150- to 100-kDa serum fractions. These results suggest that 1) functional alpha-subunit is present in TP serum; 2) intact as well as smaller fragments of IGFBP-3 are present in the big complex of TP serum and are able to bind IGF and complex with alpha-subunit; and 3) IGFBP-3 in TP serum has reduced binding to [125I]IGF-I, but not to [125I]IGF-II.