Leaves of 26 grass, herb, shrub and tree species were collected from mesotrophic grasslands to assess natural variability in bulk, fatty acid and monosaccharide δ 13C values under different grazing management (cattle- or deer-grazed) on three sample dates (May, July and October) such that interspecific and spatiotemporal variations in whole leaf tissues and compound-specific δ 13C values could be determined. The total mean leaf bulk δ 13C value for plants was −28.9‰ with a range of values spanning 7.5‰. Significant interspecific variation between bulk leaf δ 13C values was only determined in October ( P = <0.001) when δ 13C values of the leaf tissues from both sites was on average 1.5‰ depleted compared to during July and May. Samples from May were significantly different between fields ( P = 0.03) indicating an effect from deer- or cattle-grazing in young leaves. The average individual monosaccharide δ 13C value was 0.8‰ higher compared with whole leaf tissues. Monosaccharides were the most abundant components of leaf biomass, i.e. arabinose, xylose, mannose, galactose and glucose, and therefore, fluctuations in their individual δ 13C values had a major influence on bulk δ 13C values. An average depletion of ca. 1‰ in the bulk δ 13C values of leaves from the deer-grazed field compared to the cattle-grazed field could be explained by a general depletion of 1.1‰ in glucose δ 13C values, as glucose constituted >50% total leaf monosaccharides. In October, δ 13C values of all monosaccharides varied between species, with significant variation in δ 13C values of mannose and glucose in July, and mannose in May. This provided an explanation for the noted variability in the tissue bulk δ 13C values observed in October 1999. The fatty acids C 16:0, C 18:2 and C 18:3 were highly abundant in all plant species. Fatty acid δ 13C values were lower than those of bulk leaf tissues; average values of −37.4‰ (C 16:0), −37.0‰ (C 18:2) and −36.5‰ (C 18:3) were determined. There was significant interspecific variation in the δ 13C values of all individual fatty acids during October and July, but only for C 18:2 in May ( P = <0.05). This indicated that seasonal trends observed in the δ 13C values of individual fatty acids were inherited from the isotopic composition of primary photosynthate. However, although wide diversity in δ 13C values of grassland plants ascribed to grazing management, interspecific and spatiotemporal influences was revealed, significant trends ( P = <0.0001) for fatty acid and monosaccharide δ 13C values: δ 13C 16:0 < δ 13C 18:2 < δ 13C 18:3 and δ 13C arabinose > δ 13C xylose > δ 13C glucose > δ 13C galactose, respectively, previously described, appear consistent across a wide range of species at different times of the year in fields under different grazing regimes.