The stable carbon isotopic composition (expressed as δ13C) of herbivore remains is commonly used to reconstruct past changes in the relative abundance of C4 versus C3 grass biomass (C4 relative abundance). However, the strength of the relationship between herbivore δ13C and C4 relative abundance in extant ecosystems has not been thoroughly examined. We determined sources of variation in δ13C of bone collagen and tooth enamel of kangaroos (Macropus spp.) collected throughout Australia by measuring δ13C of bone collagen (779 individuals) and tooth enamel (694 individuals). An index of seasonal water availability, i.e. the distribution of rainfall in the C4 versus C3 growing seasons, was used as a proxy for C4 relative abundance, and this variable explained a large proportion of the variation in both collagen δ13C (68%) and enamel δ13C (68%). These figures increased to 78% and 77%, respectively, when differences between kangaroo species were accounted for. Vegetation characteristics, such as woodiness and the presence of an open forest canopy, had no effect on collagen or enamel δ13C. While there was no relationship between collagen δ13C and kangaroo age at death, tooth enamel produced later in life, following weaning, was enriched in 13C by 3.5‰ relative to enamel produced prior to weaning. From the observed relationships between seasonal water availability and collagen and enamel δ13C, enrichment factors (ε*) for collagen-diet and enamel-diet (post-weaning) were estimated to be 5.2‰ ± 0.5 (95% CI) and 11.7‰ ± 0.6 (95% CI), respectively. The findings of this study confirm that at a continental scale, collagen and enamel δ13C of a group of large herbivores closely reflect C4 relative abundance. This validates a fundamental assumption underpinning the use of isotopic analysis of herbivore remains to reconstruct changes in C4 relative abundance. © 2007 Elsevier Ltd. All rights reserved.