We report a sensitive fluorophotometric enzyme immunoassay (EIA) for measuring thyroxine (T4) in dried blood samples on filter paper. Anti-T4 IgG (rabbit) and the T4-horseradish peroxidase conjugate of the Enzymun Test T4 (Boehringer) were used. Bound and free fractions of the enzyme conjugate were separated by the antibody solid phase method, using anti-T4 IgG-coated bead. The enzyme activity of the bound fraction was determined fluorophotometrically at excitation and emission wavelengths of 320 and 405 nm, respectively, using 3-(p-hydroxyphenyl)-propionic acid and hydrogen peroxide as substrate. Standard curves between 1.7 and 22μg T4/dl were prepared from T4 standards of dried blood spots. The intra- and inter-assay coefficients were 4.9-13.7% and 13.7-19.5%, respectively. The present EIA method was used in preliminary mass-screening tests for T4.