The process of evaporation results in the fractionation of water isotopes such that the lighter 16O isotope preferentially escapes the gas phase leaving the heavier 18O isotope to accumulate at the sites of evaporation. This applies to transpiration from a leaf with the degree of fractionation dependent on a number of environmental and physiological factors that are well understood. Nevertheless, the 18O enrichment of bulk leaf water is often less than that predicted for the sites of evaporation. The advection of less enriched water in the transpiration stream has been suggested to limit the back diffusion of enriched evaporative site water (Péclet effect); however, evidence for this effect has been varied. In sampling across a range of species with different vein densities and saturated water contents, we demonstrate the importance of accounting for the relative ‘pool’ sizes of the vascular and mesophyll water for the interpretation of a Péclet effect. Further, we provide strong evidence for a Péclet signal within the xylem that if unaccounted for can lead to confounding of the estimated enrichment within the mesophyll water. This has important implications for understanding variation in the effective path length of the mesophyll and hence potentially the δ18O of organic matter.