The objective of this study was to assess the feasibility of screening for cholestatic hepatobiliary disease (CHD) and extrahepatic biliary atresia (EHBA) by using tandem mass spectrometry (TMS) to measure conjugated bile acids in dried blood spots obtained from newborn infants at 7 to 10 days of age for the Guthrie test. Unused blood spots from the Guthrie test were retrieved for children presenting with CHD and from the two cards stored on either side of each card from an index child. Concentrations of glycodihydroxycholanoates (GD), glycotrihydroxycholanoates (GT), taurodihydroxycholanoates (TD) and taurotrihydroxycholanoates (TT) measured by TMS in the two groups were compared. Mean concentrations of all four bile acid species were significantly raised in children with CHD and EHBA compared with the unaffected children (P 0.0001). Of 177 children with CHD, 104 (59%) had a total bile acid (TBA) concentration 33 umol/L (97.5th centile value for comparison group). Of the 61 with EHBA, 47 (77%) had TBA concentrations 33 umol/L. Receiver operator curves were plotted to determine which parameter would best predict the cases of CHD and EHBA. The sensitivity and specificity at a selection of cut off values for each bile acid species and for TBA concentration for the detection of both conditions were calculated. Taurotrihydroxycholanoates and TBA concentrations were the best predictors of both conditions. For CHD a cut off level of TBA concentration of 30 umol/L gave a sensitivity of 62% and a specificity of 96%. The corresponding values for EHBA were 79% and 96%. Although most infants with EHBA and other forms of CHD had significantly raised concentrations of conjugated bile acids at 7 to 10 days of life, the separation between the concentrations in these infants and the general population was insufficient to make mass screening for CHD a feasible option with this method alone.