In an investigation of the role of trace elements in gallstone formation and of minor and trace elements in gallstone and bile, samples from five patients were analysed using three different techniques for cross-verification: NAA, PIXE, and XRF. In addition, certified standards of bovine liver and oyster tissue from the NBS, Washington, were re-analysed using the different techniques to confirm the accuracy of the experimental procedures. NAA of five gallstones was used for the quantitative analysis of 19 elements with Z greater than 11. The concentrations of Cl, K + Ca, Mn, Fe and Cu + Zn were determined by PIXE and those of Mg, P, S, K and Ca by XRF. In most cases the concentrations were in the range of a few PPB to a few hundred PPM. The spatial distribution of the component elements was obtained using XRF with a scanning electron microscope and the results demonstrated that calcium was present centrally in all the stones. The analysis of gallbladder bile from four patients showed that the calcium concentration was much greater than normal, indicating that calcium and other elements play an important role in stone formation. Infrared spectroscopy showed that the calcium salts were present in gallstones in three compound forms: calcium carbonate, calcium phosphate and calcium bilirubinate. Reasons for the cause of calcium precipitation are discussed.