The 18O content of leaf water strongly influences the 18O contents of atmospheric CO2 and O2. The 18O signatures of these atmospheric gases, in turn, emerge as important indicators of large-scale gas exchange processes. Better understanding of the factors that influence the isotopic composition of leaf water is still required, however, for the quantitative utilization of these tracers. The 18O enrichment of leaf water relative to local meteoric water, is known to reflect climatic conditions. Less is known about the extent variations in the 18O content of leaf water are influenced by nonclimatic, species-specific characteristics. In a collection of 90 plant species from all continents grown under the same climatic conditions in the Jerusalem Botanical Garden we observed variations of about 9‰ in the δ18O values of stem water, δs, and of about 14‰ in the mid-day δ18O enrichment of bulk leaf water, δLW–δs. Differences between δ18O values predicted by a conventional evaporation model, δM, and δLW ranged between – 3.3‰ and + 11.8‰. The δ18O values of water in the chloroplasts (δch) in leaves of 10 selected plants were estimated from on-line CO2 discrimination measurements. Although much uncertainty is still involved in these estimates, the results indicated that δch can significantly deviate from δM in species with high leaf peclet number. The δ18O values of bulk leaf water significantly correlated with δ18O values of leaf cellulose (directly) and with instantaneous water use efficiency (A/E, inversely). Differences in isotopic characteristics among conventionally defined vegetation types were not significant, except for conifers that significantly differed from shrubs in δ18O and δ13C values of cellulose and in their peclet numbers, and from deciduous woodland species in their δ18O and δ13C values of cellulose. The results indicated that predictions of the δ18O values of leaf water (δLW, δM and δch) could be improved by considering plant species-specific characteristics.