Despite the high relevance of anaerobic ammonia oxidation (anammox) for reactive nitrogen removal from marine systems, its relative importance compared to denitrification has only rarely been studied in aquifers. Comparative investigations were carried out in two superimposed aquifers in the Hainich region (Thuringia, Germany) (i) to evaluate the genetic potential for anammox and denitrification in groundwater (ii) to assess the potential relevance of anammox versus denitrification by activity measurements and (iii) to investigate the groundwater-originated consortiums capacity to use inorganic electron donor for complete denitrification. Groundwater samples were taken from eight groundwater wells differed in oxygen availabilty. Abundances of nitrite reductase genes (nirS) for denitrifiers as determined by quantitative PCR ranged from 2.6 x 103 to 8.2 x 107 genes L-1 groundwater while abundances of hydrazine synthase genes (hzsA) indicative of anammox bacteria ranged from 6.9 x 102 to 2.1 x 107 genes L-1 groundwater. Functional gene transcript analysis and ladderane phospholipid provided further evidence for the presence of active anammox bacteria, mostly related to Candidatus Brocadia fulgida. Despite similar estimated abundances of denitrifiers and anammox bacteria, anammox rates (4.7 nmol N2 L-1 d-1) were about eight times higher than denitrification rates (0.6 nmol N2 L-1 d-1). Moreover, a chemolithoautotrophic denitrifying consortium was maintained in bicarbonate buffered thiosulfate-nitrate medium under argon atmosphere along with H2 and CO2 (3% headspace volume). This consortium could oxidize S2O32- and H2 completely while simultaneously reducing nitrate to nitrite and further on to dinitrogen gas, confirmed using Raman gas spectroscopy, following the conversion of 15N-nitrate to 30N2. These findings suggest a high relevance of chemolithoautotrophic processes for reactive nitrogen removal in oligotrophic limestone aquifers at Hainich, Germany.