The purposes of this study were to reevaluate age-related changes in the concentration of serum free thyroxine (T4) between early infancy and adulthood, and to study the relationship of serum thyrotropin to these changes in free T4. Five hundred thirty-six healthy infants, children, adolescents, and adults between 1 day and 45 years of age were studied. Serum free T4 was determined by direct equilibrium dialysis, thyrotropin by a sensitive immunoassay, and total T4 by radioimmunoassay. Free T4 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 T4 concentrations (mean +/- SEM) were 48.1 +/- 1.5, 20.3 +/- 0.6, 17.0 +/- 0.4, 19.9 +/- 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 T4 were complex, indicating age-related changes in pituitary thyroid regulation. No correlation existed between concentrations of free T4 and total T4 after the first 13 days of life (r2 < 0.01). We conclude that (1) pediatric free T4 concentrations and free T4-thyrotropin relationships differ from those in adults and (2) age-related changes in total T4 do not indicate age-related changes in either free T4 or thyroid function, after 13 days of age.