Proteolytic activity directed against insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is found in human pregnancy serum. We have investigated changes in this protease activity in pregnancies in which the fetus is small for gestational age and in multiple pregnancies. Maternal serum was obtained from 18 singleton pregnancies at 27 weeks gestational age (GA), and matched fetal serum was collected by cordocentesis. Fetuses were appropriate for GA (AGA; n = 6), small for gestational age (SGA) with evidence of uteroplacental insufficiency (UPI; starved SGA; n = 6), or SGA without UPI (nonstarved SGA; n = 6). In a second study, serum was obtained from women with singleton (n = 10), twin (n = 10), and higher multiple pregnancies (n = 10) at 9 weeks GA. All women with more than three fetuses underwent embryo reduction to 2 fetuses before 15 weeks gestation, when a second serum sample was obtained. Circulating IGF-I and IGF-II were measured by RIA, and IGFBP-3 was measured by both RIA and immunoradiometric assay. IGFBP-3 protease activity was assessed by Western ligand blotting after incubation with a normal nonpregnancy sera pool, immunoblotting, and specific protease assay. In the growth study, circulating maternal IGF-I and IGFBP-3 levels were not different in the three groups, but fetal IGF-I and IGFBP-3 levels were significantly lower in the UPI fetuses (IGF-I, 6.9 +/- 0.5 micrograms/L; IGFBP-3, 547 +/- 70 micrograms/L) than in either the nonstarved SGA fetuses (IGF-I, 27.8 +/- 6.3; IGFBP-3, 769 +/- 41 micrograms/L; P < 0.01) or the AGA fetuses (IGF-I, 39.4 +/- 3.4; IGFBP-3, 872 +/- 91 micrograms/L; P < 0.01). Maternal serum IGFBP-3 protease activity, measured by protease using [125I]IGFBP-3 as substrate, was increased in pregnancies complicated by UPI compared with GA-matched pregnancies in which the fetus was AGA or nonstarved SGA. No significant fetal serum protease activity was demonstrated. In the multiple pregnancies, IGFBP-3 rose significantly from 9-15 weeks GA in singleton (P = 0.005), twin (P = 0.004), and multiple (P = 0.007) pregnancies, and levels were higher in mothers of multiple pregnancies than in those of twin (P < 0.05) or singleton (P < 0.01) pregnancies at both 9 and 15 weeks GA. IGF-I levels were not different in the three groups and did not significantly increase between 9-15 weeks GA.(ABSTRACT TRUNCATED AT 400 WORDS)