The purposes of this study were to reevaluate age-related changes in the concentration of serum free thyroxine (T 4) between early infancy and adulthood, and to study the relationship of serum thyrotropin to these changes in free T 4. Five hundred thirty-six healthy infants, children, adolescents, and adults between 1 day and 45 years of age were studied. Serum free T 4 was determined by direct equilibrium dialysis, thyrotropin by a sensitive immunoassay, and total T 4 by radioimmunoassay. Free T 4 concentrations were age dependent and differed significantly ( p<0.001) at 1 to 4 days, 2 to 20 weeks, 5 to 24 months, 2 to 7 years, 8 to 20 years, and 21 to 45 years of age. Corresponding free T 4 concentrations (mean±SEM) were 48.1±1.5, 20.3±0.6, 17.0±0.4, 19.9±0.4, 17.0±0.4, and 21.2±0.4 pmol/L (3.74±0.12, 1.58±0.05, 1.32±0.03, 1.55±0.03, 1.32±0.03, and 1.65±0.03 ng/dl), respectively. Age-related reference ranges were determined. Changes in the relationship between serum thyrotropin and free T 4 were complex, indicating age-related changes in pituitary thyroid regulation. No correlation existed between concentrations of free T 4 and total T 4 after the first 13 days of life ( r 2<0.01). We conclude that (1) pediatric free T 4 concentrations and free T 4-thyrotropin relationships differ from those in adults and (2) age-related changes in total T 4 do not indicate age-related changes in either free T 4 or thyroid function, after 13 days of age.