Glucose, insulin, C peptide, and insulin antibody concentrations were measured in amniotic fluid collected under basal conditions and 2 hours after an arginine challenge from 61 insulin-treated diabetic women (12 basal and 49 after arginine challenge) and 31 nondiabetic pregnant women in late gestation (23 basal and eight after arginine challenge). The insulin, C peptide, and glucose concentrations were significantly higher in diabetic pregnant women than in nondiabetic pregnant women in each case. In the amniotic fluid obtained after arginine challenge in diabetic pregnant women, C peptide concentration was correlated with both insulin concentration (r = 0.61) and birth weight (r = 0.53). The insulin and C peptide concentrations were significantly higher (p less than 0.025) in samples from diabetic pregnancies associated with fetal morbidity than from diabetic pregnancies without fetal morbidity. Basal amniotic fluid insulin and C peptide concentrations were slightly greater in overweight infants of diabetic mothers compared to those of normal weight, whereas the differences for insulin and C peptide concentrations in the amniotic fluid obtained after arginine challenge were highly significant (p less than 0.0125 and p less than 0.0005, respectively). Finally insulin and C peptide concentrations in the amniotic fluid obtained after arginine challenge in diabetic pregnant women showed a correlation with maternal metabolic control but not with the degree (White classification) of maternal diabetes. No or negligible interference of insulin antibody in the radioimmunoassay of insulin in amniotic fluid was observed.