We have conducted a series of experiments to determine the effects different diets, including the addition of unsaturated fatty acids, had on isotopic signatures of CH4 emissions from dairy cattle. Measurements of emitted Δ13CH4, Δ13CO2, and ΔD-CH4 were made on gases collected over a 24-hour period from animal chambers containing individual Holstein cows. Some measurements of ΔD-H2O from samples collected directly from the rumen were also made. We observed variation in Δ13CH4 values with time after feeding and a correlation in Δ13C between emitted gases and diet. The average Δ13CH4 value, which includes all samples of emitted gases, was -70.6¿4.9? (n=57). Measurements of ΔD-CH4 over the 24-hour sampling period had an average value of -357.8¿15.0? (n=56). These ΔD measurements are among the lightest reported for CH4 produced by cattle. Our cattle data indicate that hydrogen incorporated into the CH4 produced by CO2 reduction in high H2 concentration environments is fractionated to a greater degree than that incorporated in systems with relatively low H2 conditions, such as wetlands. Our results support bacterial studies that have demonstrated large hydrogen fractionation in high H2 concentration systems during methanogenesis. ¿ 2001 American Geophysical Uni