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The isotopic (²³⁴U/²³⁸U, ²H, ¹⁸O) and chemical composition of groundwater on the right bank of the Volga River along the middle reach (European Russia) was studied down to a depth of 400 m. These data allow diagnosis of the presence of a three-component mixture. The first component is modern/young fresh recharge water of the Holocene age. It has the isotopic composition of water δ¹⁸O → −12.9 ‰ and δ²H → −90 ‰, close to modern precipitations, and the equilibrium isotopic composition of uranium ²³⁴U/²³⁸U → 1 (by activity). The second component is slightly salted water of the late or postglacial period with δ¹⁸O → −17.0 ‰ and δ²H → −119 ‰, and a small excess of uranium-234 ²³⁴U/²³⁸U ≈ 4. The third component is meltwater formed as result of permafrost thawing. It is brackish water with δ¹⁸O ≈ −15.0 ‰ and δ²H ≈ −110 ‰, and a maximum excess of uranium-234 ²³⁴U/²³⁸U ≈ 15.7. The salinity of this water is associated with an increase of the SO₄²⁻, Ca²⁺ and Na⁺ content, and this may be due to the presence of gypsum in water-bearing sediments, because the solubility of sulfates increases at near-zero temperature. We explain the huge excess of uranium-234 by its accumulation in the mineral lattice during the glacial age and quick leaching after thawing of permafrost.