The clinical value of free thyroxine (FT(4)) and free triiodothyronine (FT(3)) analysis depends on the reference intervals with which they are compared. We determined age- and sex-specific reference intervals for neonates, infants, and children 0-18 years of age for FT(4) and FT(3) using tandem mass spectrometry.Reference intervals were calculated for serum FT(4) (n = 1426) and FT(3) (n = 1107) obtained from healthy children between January 1, 2008, and June 30, 2008, from Children's National Medical Center and Georgetown University Medical Center Bioanalytical Core Laboratory, Washington, DC. Serum samples were analyzed using isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with deuterium-labeled internal standards.FT(4) reference intervals were very similar for males and females of all ages and ranged between 1.3 and 2.4 ng/dL for children 1 to 18 years old. FT(4) reference intervals for 1- to 12-month-old infants were 1.3-2.8 ng/dL. These 2.5 to 97.5 percentile intervals were much tighter than reference intervals obtained using immunoassay platforms 0.48-2.78 ng/dL for males and 0.85-2.09 ng/dL for females. Similarly, FT(3) intervals were consistent and similar for males and females and for all ages, ranging between 1.5 pg/mL and approximately 6.0 pg/mL for children 1 month of age to 18 years old.This is the first study to provide pediatric reference intervals of FT(4) and FT(3) for children from birth to 18 years of age using LC/MS/MS. Analysis using LC/MS/MS provides more specific quantification of thyroid hormones. A comparison of the ultrafiltration tandem mass spectrometric method with equilibrium dialysis showed very good correlation.