Our previous studies suggested that assay of immunoreactive trypsin (IRT) in dried blood spots might be a valuable neonatal screening test for cystic fibrosis (CF). We have developed a convenient, sensitive, human trypsin radioimmunoassay, which uses a 3-mm diameter disc of dried blood. The molecular species assayed in blood is trypsinogen. A retrospective study of 24 known cases of CF and appropriate controls confirmed that an elevated blood level of IRT is characteristic of all newborn CF infants, whether or not they have residual exocrine pancreatic function. IRT levels did, however, decline with time of sample storage. Guthrie cards from 5040 newborns were prospectively assayed: 2% of tests were reassayed because of elevated IRT. Thirty-three second samples (0.67% of the total) were requested, 32 were received, and 31 had normal IRT values. The baby with an elevated result had no clinical symptoms of CF at one month of age, normal stool trypsin activity, but 2 sweat tests gave grossly abnormal results. In contrast, a second infant in whom CF was diagnosed around the same time, had a similar 5-day blood spot IRT value, but severe clinical symptoms of CF and no stool trypsin activity. No false negatives are yet known. We conclude that blood spot IRT assay is a reliable and convenient neonatal screening test for CF.