Abstract: The invention relates to a method of separating isotopes from a gas mixture, of once ionized uranium the isotopes being separated by the combined effect of photon irradiation by laser light followed by Penning ionization of ions selectively excited by the laser light.According to the invention a gas of once ionized uranium isotopes is irradiated with laser light made up of photons having an energy which resonates with an excitation level of an isotope of the gas mixture. Metastable atoms or molecules are simultaneously introduced into the mixture and have an excitation energy which is less than the energy for a further ionization of the constituents of the mixture and greater than the energy gap between the energy for a further ionization of the isotope excited by the laser light and the energy of the excitation level of the same isotope.