We describe a highly sensitive and precise radioimmunoassay for thyrotropin in dried blood spots on filter paper cards. In a screening program for congenital hypothyroidism, blood-spot thyrotropin concentrations are measured in infants whose blood-spot thyroxine concentrations are in the lower 10%, and this strategy has reduced the recall rate from 1.7% (thyroxine assay alone) to 0.17%. Thyrotropin assay samples consist of discs 4.5-mm in diameter, containing about 6 microL of blood, punched from blood spots. By appropriate attention to assay conditions, a mean least-detectable thyrotropin concentration equivalent to 2.5 milliunits/L plasma has been achieved. Concomitant measurement of thyrotropin by plasma and blood-spot assays in 91 subjects yielded a Spearman rank correlation coefficient of 0.9732. An analysis of variance of the distribution volume of thyrotropin in blood spots and a covariance analysis of factors affecting blood-spot thyroxine results are presented.