Nitrogen is the most onerous nutrient in fertilization and required in great amount for many crops, especially the coffee plant. Normally, this nutrient is present in the soil in great amounts, but the mineral form is generally low. The natural ecosystem has continuous loss of N, not just by the plant absorption, but also by leaching and other types of losses. Nitrogen assimilation is a vital process to control the growth and the development of plants and has significant effects on yield. The route of nitrate assimilation in higher plants involves two sequential periods: the conversion of nitrate to ammonia, mediated by Nitrate Reductase (NR), which reduces nitrate to nitrite, and for the nitrite reductase, that converts nitrite to ammonia. Ammonia is also assimilated in the amino acids glutamine and glutamate, which serves to transport organic nitrogen from sources to sinks. The study was developed in Sud Mennucci county (northwest of São Paulo State, Brazil) in order to evaluate the effect of different rates and times of application of nitrogen on nitrate reductase and to establish correlations with yield. The experiment was developed in the agricultural season of 2003/2004 in field conditions with the Coffee Cultivar Catuaí Vermelho (five years old), 3,0m of row spacing and 1,0m between plants. The experimental design was completely randomized, in a factorial system 5x3, with five nitrogen (urea) rates (0, 50, 150, 250, and 350 kg ha^-1) and three application times (a single application in December; two applications: parceled in November and January and three applications: parceled in November, December and January). Response of the activity of the enzyme nitrate reductase to nitrogen fertilization, and affects on yield were measured. The activity of the NR is positively correlated with the nitrogen content in the leaves and the yield. The apical region of the coffee tree overestimates the activity of the NR, while the basal region shows contrary behavior. The adjusted place for the leaf collection to laboratory analysis of NR is the middle of the plant. The activity of the NR is a good parameter to estimate the yield of the coffee tree. Nitrogen levels of 350 kg ha^-1 provides higher values of nitrogen content in the leaves. The level of 210 kg of N ha^-1 provides highest yield and adjusted values of nitrogen content in the leaves.