In a variety of physio-pathological conditions, but also in the normal state, calcium-dependent serine protease(s) partially proteolyze proportions of circulating insulin-like growth factor binding protein-3 (IGFBP-3). This occurs without disrupting the 150-kilodalton (kDa) complexes in which IGFBP-3 carries of most of the IGF-I and IGF-II in serum. In this work we show that the 150-kDa complex is functionally altered during pregnancy, which is when the largest proportion of IGFBP-3 is proteolyzed. After preincubation at 4 C with [125I]IGF-I or -II with or without 1 microgram unlabeled IGF-I or -II, pooled plasma samples were gel filtered at pH 7.4. Larger proportions of labeled IGFs were found in the 150-kDa complexes of the third trimester pregnancy plasma pool than in those of the normal plasma pool, suggesting increased binding capacity. Nevertheless, competitive binding experiments using [125I]IGF-I and -II and IGFBP-3 preparations from each of the plasma pools showed that the competitive potencies of IGF-II and especially IGF-I were lower in pregnancy IGFBP-3 than in normal IGFBP-3. Scatchard analysis revealed a 2-fold loss of affinity for IGF-II and a 10-fold loss for IGF-I compared with that for normal plasma IGFBP-3. In studies at 37 C of the kinetics of dissociation of [125I]IGF-I and -II bound to IGFBP-3, IGF-II was dissociated 6 times faster, and IGF-I 10 times faster from pregnancy plasma IGFBP-3 than from normal plasma IGFBP-3. After incubation of individual plasma samples at 37 C and gel filtration at room temperature (in the presence of EDTA), IGFs were assayed in the three circulating pools (150-kDa and 40-kDa complexes and free IGFs), revealing a redistribution of pregnancy plasma IGFs. The proportion of total IGF-I in free form was nearly three times greater in pregnancy than in normal plasma (11.4% vs. 4.1%, P < 0.005), whereas that of free IGF-II was slightly smaller (1.5% vs. 2%). In the 150-kDa complexes, the proportion of total IGF-I was significantly smaller in pregnancy than in normal plasma, and that of IGF-II was greater. In the 40-kDa complexes, the proportion of total IGF-II was significantly smaller. The mean ratios of molar concentrations of free IGF-I/IGF-II were 0.43 in normal plasma and 2.23 in pregnancy plasma (P < 0.005).(ABSTRACT TRUNCATED AT 400 WORDS)