1. A procedure for measuring methane production by rumen contents incubated anaerobically in vitro is described. Assessments of methane production in vivo, in both sheep and cattle, were made by withdrawal of rumen contents and measuring their capacity to produce methane in vitro.

2. Many members of a series of 6-substituted 2,4-bis (trichloromethyl)-benzo[1,3]dioxins were potent inhibitors of methanogenesis by rumen contents in vitro. The most potent compound inhibited methane production by 70% or more at a concentration of 1 μg/ml (approximately 2.5 μmol/l).

3. Two compounds, namely the 6-carboxylic acid (IC1 13409) and the 6-carboxamide (ICI 43586), caused a large inhibition of methanogenesis sustained for many hours, following a single intrarumen injection in sheep or cattle. Inhibition was maintained for long periods by single daily dosing directly into the rumen or by dietary administration.

4. In a 28-week growth trial in beef cattle inclusion of ICI 13409 in the concentrate element of the diet, at a level of 6 mg/kg body-weight, improved live-weight gam by 8·0% (P < 0·05) with respect to untreated animals whilst reducing food intake by 5·0% (P < 0·05). Smaller and not statistically-significant effects were seen with this compound at 3 mg/kg body-weight and with the antibiotic monensin(Romensin; ElancoPLC). All treatments significantly improved the retention of dietary energy into the carcass, offal and intestinal tracts of the trial animals and significantly reduced the quantity of methane eructed into expired gases.