Hydrogeochemical investigations and spatial variations studies on the distribution of water isotopes and radiocarbon in the groundwater of Žitný Island (Rye Island) were carried out. Žitný Island represents the largest groundwater reservoir in Central Europe (about 10 Gm3). The chemical composition of the groundwater of Žitný Island depends mainly on the chemical composition of Danube water, as well as on the length of its infiltration from the Danube River. The groundwater is characterized by potamogenic mineralization, and its chemical composition is influenced by anthropogenic contamination. Sub-surface water profiles showed enriched δ18O levels up to around 20 m water depth, and depleted values for deeper waters. The observed isotopic composition of the groundwater is similar to Danube water, suggesting that the Danube River is the main source of the Žitný Island groundwater. The core of the sub-surface 14C profile represents contemporary groundwater with 14C values above 80 pMC.