Background: This study was designed to establish a reference interval for sweat chloride for infants without evidence of cystic fibrosis (CF), aged between 5 wk and 6 wk, a time when sweat testing is an integral part of newborn screening for CF. In addition, we compared the gold standard method of sweat testing (quantitative pilocarpine iontophoresis [QPIT, coulometry]) with an emerging methodology (MacroductTM [ISE]). Methods: This was a prospective study on healthy infants at 5–6 wk of age. Sweat collection was undertaken at home on both outer thigh areas using two methods (QPIT and MacroductTM). The order of testing was randomly assigned. Filter paper samples (QPIT) were analysed using flame photometry and coulometry. MacroductTM samples were analysed using ion-selective electrodes (ISE, Abbott Architect c8000, UK). Results: Insufficient sweat was collected on 28 occasions with the QPIT (coulometry) method and on 31 with the MacroductTM (ISE) capillary system. We achieved a 92% success rate in undertaking two sweat collections consecutively (n = 177). Sweat chloride concentrations were normally distributed with excellent limits of agreement between the two methods of sweat collection and analysis (n = 150). Median (IQR) sweat chloride was 11.2 mmol/L (8–13) with QPIT (coulometry) method with a 99.5th centile (n = 165) of 24 mmol/L. Conclusion: The MacroductTM (ISE) capillary sweat collection system is valid in this age group. Sweat chloride concentrations above 30 mmol/L should prompt assessment in a specialist CF centre.