Congenital hypothyroidism (CH) is one of the most common preventable causes of mental retardation. Newborn screening and thyroid therapy started within 2 weeks of age can normalize the cognitive and motor impairment caused by lack of thyroid hormone during the early postnatal phase of brain development. The availability of assays to determine thyroxin (T4) and thyroid stimulating hormone has allowed the establishment of newborn screening programs for CH. The Immunoassay Center (Cuba) developed the T4 Neonatal UMELISA(r) to determine neonatal T4 levels in dried blood on filter paper using 5 mm discs of samples, standards and controls. The number of diseases which can be diagnosed in Neonatal Screening Programs has increased, so it is necessary to make the maximum use of blood samples collected on filter paper. This work shows the standardization of the T4 Neonatal UMELISA(r) modified in order to use 3 mm discs. The assay was completed in 3 1/2 hours, with a measuring range of 25-400 nmol/L. The intra- and inter-assay coefficients of variation (CV) were 6-10 % and 7-12 % respectively, depending on the T4 concentrations. The recovery ranged from 91.8-115.1 %. The modified assay showed high Pearson correlation (r = 0.877) and concordance correlation (?c = 0.867) with the T4 Neonatal UMELISA(r). The performed modifications do not affect the sensitivity, precision and accuracy of the assay, permitting a more rational use of newborn blood samples and the possibility to increase the number of diseases included in the newborn screening programs.