Methane emissions are widely recognized as one of the major factors contributing to global climate change. Methane is produced by livestock as part of their normal digestive process and represents a significant source of greenhouse gases particularly in New Zealand. It also represents a loss of dietary energy to animals. Methanogens, a group of archaeal microorganisms, are the principal methane forming organisms in the rumen. They are evolutionarily and biochemically distinct from bacteria, humans and ruminant animals. They possess a number of specialized biochemical pathways, including those for methanogenesis, cofactor biosynthesis, and lipid synthesis. We have exploited these distinctions to specifically target many of their enzymes in order to develop small molecules as inhibitors capable of mitigating ruminant methane emissions and, possibly, improving production. Over 300 methanogen enzymes were targeted for cloning and expression of enzymes in Escherichia coli, 17 enzyme structures determined and 8 enzyme assays developed. Five of the structures and six enzyme assays have been used for high-throughput screening of compound libraries. This screening has led to the identification of a number of novel inhibitory compounds that are active in rumen fluid-based in vitro experiments and that are being tested in animal trials.