This paper presents a computer algorithm for triple radionuclide subtraction studies and its application to preoperative localization of enlarged parathyroid glands. In the clinical examination procedure, 131I-toluidine blue was used as the principal radionuclide, 99mTcO4 and 113mIn were used to obtain additional images of the thyroid and the blood distribution respectively. A scintillation camera with a pinhole collimator and connected to a digital data aquisition system was used to record the images. Subtraction of uniform background, thyroid and blood contributions to the principal image is done automatically in the computer program. The results from a clinical study is used to illustrate the method.