OBJECTIVE To compare 17-hydroxyprogesterone (17-OHP) levels measured by quantitative serum radioimmunoassay (RIA), including an extraction step, and by screening fluoroimmunoassay (FIA) on blood spots in preterm infants. METHODS Subjects were 39 healthy infants born at less than 31 weeks' gestational age. Each infant had weekly blood sampling, and RIA and FIA were performed on each sample. RESULTS Two hundred twenty-seven samples were taken at 28 to 41 weeks' postconceptional age. Mean +/- SD 17-OHP measured by RIA was 11.4 +/- 11.1 nmol/L (0.4 +/- 0.4 micrograms/dL), and decreased over time. Mean +/- SD 17-OHP measured by FIA was 38.96 +/- 37.3 nmol/L, greater than 17-OHP (RIA). Log(delta FIA-RIA) was inversely related to postconceptional age (R2 = .39). CONCLUSIONS Screening FIA of blood spots overestimates levels of 17-OHP in preterm infants and should not be used to determine the likelihood of congenital adrenal hyperplasia in this population. We have abandoned FIA screening for congenital adrenal hyperplasia in infants weighing less than 1500 g.