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Applying manure reduces the requirement for mineral nitrogen (N) fertilizer use and also increases carbon (C) sequestration in soils. This study was conducted to quantify the effect of manure application on the net ecosystem carbon balance (NECB), methane (CH4) and nitrous oxide (N2O) emissions at four managed grasslands in different climatic regions in Japan. At each site, two adjoining treatments were established, one for the application of farmyard manure, and the other for the application of mineral fertilizer. Net ecosystem exchange (NEE) of carbon dioxide (CO2) was quantified using eddy covariance, and CH4 and N2O emissions were quantified using static chambers from 2005 to 2007. The manure application rate was based on the potassium demand of grasses. NECB was estimated as NEE minus C applied in manure plus harvested C. NECB in the fertilizer and manure treatments were 1.9±0.9 and -1.8±1.8 Mg C/ha/y, respectively, indicating that the manure plots gained C. Harvested C in the fertilizer and manure treatments were not significantly different (P<0.05). NEE showed a greater CO2 uptake in the fertilizer treatments (-2.4±1.1 Mg C/ha/y) than in the manure treatments (-1.6±0.7 Mg C/ha/y), but C input through manure application could compensate the shortage in NEE. The emission of CH4 was close to zero in the fertilizer and manure treatments (0.01±1.25 and -0.22±0.23 kg C/ha/y, respectively). Significant differences (P<0.05) in N2O emissions were found between the fertilizer and manure treatments (3.6±3.2 and 6.2±3.7 kg N/ha/y, respectively). The global warming potential was significantly higher in the fertilizer treatments than in the manure treatments. Results from this study suggested that the application of manure may mitigate global warming in Japanese grasslands.