Abstract Fertilization is one of the factors that can contribute to build-up of nitrates in kikuyu (Cenchrus clandestinus) pastures of the tropics. Field studies to evaluate the influence of nitrogen fertilization on yield and nitrate concentrations were conducted between December 2012 and June 2013 at the Agricultural Station of the National University at Santa Elena (Antioquia). The experimental design was a 4 x 4 block design with four replications in a monoculture paddock of kikuyu grass. Treatments were four levels of nitrogen applied as topdressing urea granules (0, 50, 100 and 200 kg/ha per plot). Whole grass samples (blades and stems) were collected at 42 days post-fertilization to mimic cattle grazing exposure. Biomass was estimated as dry matter yield per hectare and nitrates were measured by ion chromatography and expressed as parts per million (ppm) on dry matter content. No significant difference in biomass was observed between urea application rates; however, all treated plots produced more biomass than non-treated plots (mean 2.14 vs 1.12 Tons/ha, p<0,05). Mean ± SEM concentrations of NO3 for all time intervals combined increased from 369 ± 216 ppm with no fertilization, to 878 ± 881 ppm at the highest application rate. In spite of the large variation within each group, the highest NO3 level attained was 5600 ppm NO3, which are considered safe for cattle consumption. It can be concluded that nitrate levels in kikuyu pastures fertilized with 4 times the usual rate of nitrogen did not accumulate to pose a health risk in cattle; however, other factors that could also influence nitrate accumulation were not determined in this study