1. A strong relationship between the oxygen isotope composition (expressed as δ18O) of body water and relative humidity has been demonstrated for a number of mammalian herbivores with low drinking water requirements, including kangaroos. Consequently, it has been suggested that the oxygen isotope composition of preserved mammal remains may be used to reconstruct past relative humidity. Other physiological, environmental and ecological factors may also influence mammalian δ18O, thereby confounding the climatic signal, yet these factors have been rigorously examined in few taxa. 2. We examined sources of variation in the δ18O of tooth enamel, assumed to reflect δ18O of body water, of kangaroos (Macropus spp.) collected throughout Australia. 3. Relative humidity explained a large proportion of the variation in enamel δ18O, a finding that is consistent with previous studies. However, we also found a previously unreported interaction between mean annual temperature and relative humidity. At lower temperatures, the relationship between enamel δ18O and relative humidity was much steeper than at higher temperatures. 4. This may be a consequence of the Peclét effect in plant leaves, whereby high transpiration rates diminish the 18O enrichment of bulk leaf water. It is likely that this interaction is also present in other herbivores with low drinking water requirements. 5. We found little evidence that δ18O varied consistently between molars, suggesting that a 'weaning effect' is either absent or swamped by seasonal variation in precipitation δ18O. 6. We suggest that the oxygen isotope composition of preserved kangaroo remains cannot be used to reconstruct relative humidity unless ambient air temperature can be reliably estimated. © 2007 The Authors.