The study aims to determine the source of NH4+ ions in the mineral waters of the Hranice Karst. The study area is located in the eastern part of the Czech Republic, Europe. The area is known mainly for its carbon dioxide of deep origin; the gas was the factor that enabled the formation of hypogene karst, in the Palaeozoic limestones, as well as warm mineral waters. The limestones of the area are covered by Neogene (Miocene) sediments of variable thickness and lithology. Recurrent sampling was done at 36 sites. A total of 96 surface water samples, 65 borehole water samples and 96 karst water samples were assessed. Major anions, cations and the content of nitrogen and its forms were determined for all water samples. The soil types were characterised by a field pedological survey. The normalised difference vegetation index was calculated in QGIS and vegetation vitality was evaluated. Since places with remarkably low vegetation index were found to be linked to the occurrence of Miocene sandstones, they represent points of rather fast entry of rainwater into the ground. As the presence of carbon dioxide creates an anoxic setting underground, the entering nitrates are transformed into NH4+ ions. This mechanism of transformation within the nitrogen cycle explains the presence of NH4+ ions in areas with elevated CO2.