BACKGROUND: High levels of 17-hydroxyprogesterone (17-OHP) are frequently observed in premature infants without congenital adrenal hyperplasia. The purpose of this study is to set cut-off limits of 17-OHP on the basis of gestational age at birth and birth weight. METHODS: Blood spot 17-OHP concentrations were measured in 1,000 infants on the 3th day of life at Inha University Hospital. An enzyme-linked immunosorbent assay (ELISA) method (ICN Neoscreen ELISA 17-hydroxyprogesterone kit, ICN Pharmaceuticals. Inc., Japan) was used. The values obtained were analyzed with respect to birth weight and gestational age at birth in order to decide the appropriate cut-off limits in a neonatal mass screening for 21-hydroxylase deficiency. RESULTS: In the neonatal mass screening for CAH, the cut-off limits for determining the 17-OHP for recall, were decided as follows: (1) 57.65, 39.88, 33.52 ng/mL for gestational age at birth of 35 weeks or less, 36-37, and 38 weeks or more, respectively, and (2) 54.88, 43.86, 32.92 ng/mL for birth weight of 2.49 or less, 2.50-2.99, 3.00 kg or more, respectively. CONCLUSIONS: The cut-off limits on the basis of gestational age at birth and birth weight should be used in the screening for congenital adrenal hyperplasia. We believe that the false positive rate in premature infants can be reduced using this method.