BACKGROUND: While the sensitivity of newborn screening for the salt wasting form of congenital adrenal hyperplasia (CAH) is good, the positive predictive value is poor due to the high false positive rate of the immunological assays for 17-OHP. Cross-reactivity with steroid sulfates is one of the main causes for false positive results. Several approaches have been described to improve CAH screening: adjusting cut-off levels to gestational age or birth weight, and second-tier molecular genetic analysis or second-tier liquid chromatography-tandem mass spectrometry (TMS). METHODS: 17-OHP was extracted with diethyl ether from dried blood spots in order to separate 17-OHP from polar steroids (like steroid sulfates). The dried ether extracts of calibrators, controls, and patient samples were redissolved and measured with the 17-OHP test kit (Wallac). RESULTS: 760 normal, 1049 false positive, and 232 samples of confirmed cases with CAH were analysed. Mean 17-OHP values were significantly lower after extraction: Normal samples: 17.5 nmol/L vs. 3.2 nmol/L; false positive samples: 97.0 nmol/L vs. 25.9 nmol/L; CAH: 275 nmol/L vs. 205 nmol/L. With a cut-off value of 11.9 nmol/L (mean+3 SD of the normal values), 404 of the false positives turned out to be normal. Ether extraction revealed two distinct subgroups of initially false positives rather than a continuum with normal distribution of 17-OHP values. CONCLUSION: Diethyl ether extraction provided evidence for two causes of false positive results in CAH screening. It reduced the rate of false positives by about 40% without loss of sensitivity.