The objective of this study was to examine the effects of nitrate on methane production, important fermentation characteristics, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, total bacteria, and methanogens using in vitro ruminal cultures. Potential adaptation of the above microbes and persistency of nitrate to mitigate CH4 production were also evaluated. Methane production was reduced by 70% at 12 μmol ml−1 and nearly completely at ⩾24 μmol ml−1 nitrate. Production of volatile fatty acids (VFAs) was affected to different extents at different nitrate concentrations. Over a series of six consecutive cultures receiving 12 μmol ml−1nitrate, production of CH4 and VFA did not change significantly. R. albus and R. flavefaciens seemed to adapt to nitrate, while F. succinogenes and methanogens did not. Nitrate may be used in achieving persistent mitigation of CH4 production by ruminants.