An absolute method for the determination of isotopic composition of substances in multiple collector mass spectrometers was proposed. The detailed analysi of terms used in the method for the determination of the isotopic composition of uranium hexafluoride was given. Parabolic nature of occurring constant measurement error, obtained in theory, was proved by test data for 235U within a wide range of its concentrations. In order to use the method of measurements in practice, the registration of constant error, through linear discriminatory relations Δc i(c i), preliminary determined using uranium hexafluoride with standard isotopic composition, was proposed. The advantages of the developed method in comparison with the traditional analysis procedures, applied at present, were considered. © Pleiades Publishing, Ltd., 2011.