Thyroid hormone concentrations in amniotic fluid (AF) can aid in the diagnosis of fetal hypothyroidism. However, the availability of reference intervals for thyroid hormones in AF using current methods is limited. The purpose of this study was to validate the Bayer ADVIA Centaur (Bayer Healthcare, Tarrytown, NY) third-generation thyroid-stimulating hormone (TSH), total thyroxine (T4), and free thyroxine (FT4) assays for use with AF and establish reference intervals for these thyroid hormones in AF. Third-trimester AF samples were assayed for TSH, T4, and FT4. Reference intervals were calculated using nonparametric analyses. The reference intervals were as follows: TSH (n = 131), 0.04 to 0.51 microIU/mL (0.04-0.51 mIU/L), with a median of 0.10 microIU/mL (0.10 mIU/L); FT4 (n = 133), less than 0.10 to 0.77 ng/dL (1.29-9.93 pmol/L), with a median of 0.26 ng/dL (3.35 pmol/L). T4 in AF was undetectable using the Centaur assay, reinforcing the importance of validating different manufacturers' immunoassays for use with nonserum specimens. These reference intervals represent the first study performed to date using a third-generation TSH assay and a sensitive FT4 assay.