Laboratory experiments were conducted with Daphnia magna and Hyalella sp. grown on a single food source of known isotopic composition at a range of temperatures spanning the physiological optima for each species. Daphnia raised at 26.5°C were enriched in δ13C and δ15N by 3.1 and 2.8‰, respectively, relative to diet. Daphnia raised at 12.8°C were enriched 1.7 and 5.0‰ in δ13C and δ15N, respectively. Results imply a significant negative relationship between the δ13C and δ15N of primary consumers when a temperature gradient exists. Similar responses were observed for Hyalella. Results indicate a general increase in δ13C enrichment and decrease in δ15N enrichment as temperature rises. Deviations from the commonly applied isotopic enrichment values used in aquatic ecology were attributed to changes in temperature-mediated physiological rates. Field data from a variety of sources also showed a general trend toward δ13C enrichment with increasing temperature in marine and lacustrine zooplankton. Multivariate regression models demonstrated that, in oligotrophic and mesotrophic lakes, zooplankton δ13C was related to lake-specific POM δ13C, lake surface temperature and latitude. Temperature-dependent isotopic separation (enrichment) between predator and prey should be taken into consideration when interpreting the significance of isotopic differences within and among aquatic organisms and ecosystems, and when assigning organisms to food-web positions on the basis of observed isotope values. Copyright © 2003 John Wiley & Sons, Ltd.