Aiming at estimating the average N2-fixation in a pasture, ap preciating the great variability due to patchy urine and dung deposition, the in fluence of dairy cow excreta on biological N2-fixation in a perennial ryegrass–white clover mixture was studied using natural urine and dung. Application of urine as well as dung affected the N2-fixation by promoting the growth of grass and thereby the proportion of clover was significantly reduced. Also the proportion of clover-N derived from the atmosphere (pNdfa) was significantly reduced. In control plots clover dry matter constituted between 40 and 50% of the total dry matter production and the pNdfa ranged between 0.8 and 0.9. Addition of urine caused a significant increase in the grass growth rates, which was the primary reason for a decrease in proportion of clover. At the same time pNdfa decreased to 0.2–0.4 followed by an increase resulting in a total reduction of 45% in the N2-fixation in urine affected areas over a period of four months. The dung only affected the N2-fixation for a distance of up to 10 cm from the edge of the dung pats. In this border area the pNdfa decreased from 0.85 to 0.75 during one month after application followed by an increase, so that after three months there was no difference between pNdfa at 0–10 and 10–20 cm distance from the dung hill. The proportion of clover was lower in the 0–10 cm than in the 10–20 cm distance, which totally resulted in a total reduction of 20% in the N2-fixation over a period of four months in the 0–10 cm area around the dung pats. Considering the proportion of a pasture which may by affected by excreta at a stocking density of 4–6 cows ha-1, the length of the grazing period, the frequency of excretion and the area covered by individual patches, it was estimated that the N2-fixation in a grass-clover pasture would be reduced by 10–15% compared to the N2-fixation in a grass-clover sward not exposed to animal excreta.