OBJECTIVE: To examine the hypothesis that early second trimester amniotic fluid (AF) insulin concentration is elevated and later fetal growth is augmented in gravidas demonstrating later oral glucose intolerance. RESEARCH DESIGN AND METHODS: In this prospective observational cohort study, AF was sampled at 14-20 weeks' gestation in 247 subjects, and 1-h 50-g oral glucose challenge tests (GCTs) were performed at > or = 24 weeks. AF insulin was assayed by an automated immuno-chemiluminometric assay (8). Macrosomia was defined as birth weight above the 90th centile. RESULTS: AF insulin concentration (range 1.4-44.5 pmol/l) correlated positively with gestational age and maternal weight. A logistic regression analysis, adjusted for maternal age and midpregnancy weight, showed increased AF insulin multiples of gestational age-specific medians to be associated with subsequently diagnosed gestational diabetes mellitus (GDM) (OR 1.9, CI 1.3-2.4, P = 0.029). Among 60 subjects with GCT values > 7.2 mmol/l, each unit increase in AF insulin multiple of median (MOM) was associated with a threefold increase in fetal macrosomia incidence (3.1, 1.3-4.9, P = 0.048). CONCLUSIONS: An elevated AF insulin concentration at 14-20 weeks' gestation is associated with subsequently documented maternal glucose intolerance. Among gravidas with GCT values > 7.2 mmol/l, elevated early AF insulin concentration is associated with fetal macrosomia. Maternal glucose intolerance may affect fetal insulin production before 20 weeks' gestation.