Background: Newborn screening for cystic fibrosis (CF) with immunoreactive trypsinogen (IRT) and ΔF508 analysis followed by sweat testing misses some infants with CF and detects more ΔF508 carriers than expected. Some of the apparent ΔF508 carriers may be ΔF508 compound heterozygotes with normal sweat electrolyte levels. Methods: Infants identified by newborn screening with an elevated IRT level, one ΔF508 allele, and a sweat chloride level <60 mmol/L underwent CF mutation analysis, pancreatic stimulation testing, and repeat IRT analysis followed by clinical review and repeat sweat test at 12 months. Results: Over a 24-month period we identified 122 ΔF508 heterozygotes and recruited 57; 4 had borderline sweat chloride levels (40 to 60 mmol/L), 5 (8.8%, 95% CI 1.4, 16.2) had a second CF mutation (R117H), and 11 (20%, 95% CI 10, 30) had the intron 8 5T allele. Three had clinical CF at 12 months (initial sweat chloride levels: 53, 51, and 32 mmol/L). Pancreatic electrolyte secretion in the subjects with a borderline sweat chloride level was similar to that in patients with known CF. Conclusion: The excess of ΔF508 heterozygotes detected by IRT/DNA screening is associated with the presence of a second mutation or the 5T allele in some infants. Screened infants with borderline sweat chloride levels almost certainly have CF, but long-term follow-up of the infants with the genotype ΔF508/R117H and ΔF508/5T is required to determine their outcome. In the meantime, newborn screening should be confined to severe mutations associated with classic CF. (J Pediatr 2000;137:214-20)