A novel technique of lithotripsy was investigated with a mid-infrared tunable pulsed laser using difference-frequency generation (DFG). Human gallstone samples obtained from 24 patients were analyzed with their infrared absorption spectra. It was found that the principal components of the gallstones were different for the different patients and that the gallstone samples used in this research could be classified into four groups, i.e., mixed stones, calcium bilirubinate stones, cholesterol stones, and calcium carbonate stones. In addition, some gallstone samples had different compositions within the single stone. The mid-infrared laser tunable within a wavelength range of 5.5 - 10 μm was irradiated to the cholesterol stones at two different wavelengths of 6.83 and 6.03 μm, where the cholesterol stones had relatively strong and weak absorption peaks, respectively. As the result, the cholesterol stones were more efficiently ablated at the wavelength of 6.83 μm with the strong absorption peak. Therefore, it is suggested that the gallstones could be efficiently ablated by tuning the wavelength of the laser to the strong absorption peak of the gallstones. The higher efficiency of the ablation using the characteristic absorption peaks should lead to the safer treatment without damage to the surrounding normal tissues. In order to identify the composition of the gallstones in the patients, endoscopic and spectroscopic diagnosis using the DFG laser and an optical fiber probe made with two hollow optical fibers and a diamond attenuation total reflection prism should be useful. The absorption spectrum of the gallstones in the patients could be measured by measuring the energy of the DFG laser transmitted through the optical fiber probe and by scanning the wavelength of the DFG laser.