Fetal goiter can arise as a result of fetal hyper or hypothyroidism. Although this condition is rare, it can be life threatening. Detection of fetal goiter in utero is possible with the aid of ultrasound, but proper prenatal treatment depends on knowledge of hormonal status. Amniotic fluid (AF) sampling is less technically demanding and poses fewer risks to the fetus than cordocentesis for fetal serum sampling, but well-established reference ranges for AF thyroid studies are not available in the literature. We have established reference intervals for AF (TSH), total T4 (tT4), and free T4 using stored AF samples. The reference intervals were: TSH (n = 127), less than 0.1-0.5 mU/liter, with a median of 0.1 mU/liter; tT 4 (n = 129), 2.3-3.9 μg/dl (30-50 nmol/liter), with a median of 3.3 μg/dl (4 nmol/liter); and free T4 (n = 119) less than 0.4-0.7 ng/dl (5-9 pmol/liter), with a median of 0.4 ng/dl (5 pmol/liter). These intervals represent the largest study done to date on third trimester AF using automated immunoassays. A literature search of fetal goiter revealed a number of cases of hypothyroidism. Seven cases reported AF TSH concentrations (range, 1.1-28.9 mU/liter) and four reported AF tT4 concentrations [range, 0.98-1.25 μg/ml (13-16 nmol/liter)], all of which fell outside our reference intervals. These data support the use of AF to diagnose fetal hypothyroidism, reducing the need to resort to a riskier procedure such as cordocentesis.