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A repeated soil survey (1995 and 2006) on 66 ICP Forests pair plots in the Czech Republic revealed a significant relationship between modeled nitrogen deposition and nitrogen concentration in the soil. Nitrogen deposition was modeled for the years 1995, 2004 and 2006. We found a more significant relationship between deposition data in 2004 and soil data in 2006 than between deposition and soil data from the same year 2006. Concentration of total nitrogen in forest soil increased from 1995 to 2006. Forest soil showed effects of increased nitrogen input from the humus layer to around 20 cm depth of mineral soil. The occurrence and cover of nitrophilous species in the herb layer increased from 1995 to 2006 in 25% of the analyzed plots, which corresponds to the nitrogen increase in forest soil. The results suggest that nitrogen deposition still represents a threat for Czech forest ecosystems.