ABSTRACT: Although there are small amounts of nitrate in almost all aquatic environments, higher amounts have indirect toxic effects on the human body. Therefore, nitrate concentration is strictly limited in Slovak drinking water where it is restricted to 50 mg·L-1 (Government Regulation No. 496/2010 Coll.). Protection of water against nitrate pollution from agricultural sources is covered in the Council Directive 91/676/EEC (Nitrates Directive). A total of 1,717 Slovak observation sites were designated for evaluation of nitrate content in groundwater between 2008 and 2011. Although the average nitrate concentration was less than 50 mg·L-1 in 89 % of observation sites, higher values were detected at 189 monitoring stations, and most of these were in designated vulnerable zones. The highest number of monitoring sites with average nitrate concentration over 50 mg·L-1 were in Nitra, Levice, Nové Zámky, Zlaté Moravce and Bánovce nad Bebravou, where nitrogen fertilizer application ranged from 100 to 109 kg N·ha-1·yr-1. However, it is impossible to definitively exclude the impact of intra-urban municipalities on increased groundwater NO3- levels. There were 681 monitoring sites selected to assess trends of nitrate concentration in groundwater and compared in 2004 – 2007 and 2008 – 2011. Results showed (1) a decreasing trend in nitrate average contents in 34 % of evaluated sites, (2) almost 45 % had a stable trend and (3) 21 % had increasing trend. However, nitrate concentration was less than 25 mg·L-1 in many monitored sites with increasing trend. Implementation of measures laid down in Action programmes (Decree of the Ministry of Agriculture of the Slovak Republic No. 199/2008 Coll.) and the Code of Good Agricultural Practice should ensure that nitrate penetration into groundwater does not radically increase in future years.