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To evaluate the effectiveness of a second newborn screen for congenital adrenal hyperplasia (CAH) in the state of Colorado and report characteristics associated with cases identified on the first versus second screen.Colorado implemented newborn screening for CAH with 17-hydroxyprogesterone beginning August 2000. The first screening is performed within 72 hours of life and the second between 8 and 14 days of life. We compared infants diagnosed on the basis of the first versus second newborn screen.The first screen identified 29 cases of which 28 represented classical CAH. The incidence of classical CAH on the first screen was 1:24 766. The second screen identified 17 additional cases, of which 11 represented classical CAH. Combined, the incidence of classical CAH was 1:17 789. The sensitivity of the first screen was 71.79%. The false negative rate of the first screen was 28.2%. In the absence of a second screen, 1:47 824 infants would have been missed. Infants diagnosed on the first screen had higher 17-hydroxyprogesterone values compared with those diagnosed on the second screen (P = .0008).The use of a single newborn screen for CAH missed nearly 30% of classical CAH cases in Colorado. Addition of a second screen, therefore, can improve the operating characteristics of the newborn screening program.