A rapid, sensitive radioimmunoassay for thyroxine (T4) is described which requires a specimen of dried blood on filter paper. One milliliter of glycine-acetate buffer containing anti-T4 antibody, tracer T4, and sodium salicylate is added to a tube containing a 1/8-inch dot of the filter paper specimen. After incubation overnight, bound and free hormone are separated by addition of dextran-coated charcoal. Quantitation is obtained using a standard curve prepared from dots of dried blood samples with known T4 content. The dot remains in the solution throughout the procedure. Recovery of T4 is 95% and intra- and interassay coefficients of variation are both less than 10%. The mean T4 content of 983 samples from the 3-day-old infants was 189 +/- 48 pg T4/dot (mean SD). This corresponds to the T4 in 1.5 mul plasma, and thus the estimated plasma T4 in these infants is 12.6 +/- 3.2 mug T4/100 ml. Nine neonates had repeated samples in which the T4 content was lower than 2 SD below the mean. All of these infants had normal cord thyroid-stimulating hormone (TSH) concentrations and thus presumably do not have primary hypothyroidism. The method should be useful for screening neonates (and older infants), since it can be adapted for use with the punch-index machine for automated processing, no prior extraction of T4 from the dot is required before quantitation, and the small size of the sample allows repeated tests of suspicious results.