The CULTAN (Controlled Uptake Long Term Ammonium Nutrition) system is based on one-time injection of the whole dose of nitrogen required for the vegetation period. The effect of this method on yield and grain quality of winter wheat was observed in a 2-year small-plot trial at 4 different experimental sites in the Czech Republic. The experiment comprised two treatments with the total amount of nitrogen applied during fertilization of 150 kg N/ha. At the CULTAN treatment the whole dose was applied all at once using the GFI 3A injection machine (Maschinen und Antriebstechnik GmbH Güstrow), whereas at the control treatment, the dose was divided into three applications. The average grain yield of winter wheat in 2007 was 9.56 t/ha (control) and 8.78 t/ha (CULTAN); in 2008 it was 9.91 t/ha (control) and 9.63 t/ha (CULTAN). The differences in 2008 were not statistically significant. The contents of nitrogen and gluten were significantly lower at CULTAN treatment in both years. The values of falling number, Zeleny test and bulk density were generally similar at both treatments.