We developed and tested a theoretical model describing carbon isotope discrimination during photosynthesis in tree bark. Bark photosynthesis reduces losses of respired CO from the underlying stem. As a consequence, the isotopic composition of source CO and the CO concentration around the chloroplasts are quite different from those of photosynthesizing leaves. We found three lines of evidence that bark photosynthesis discriminates against C. First, in bark of Populus tremuloides, the δC of CO efflux increased from -24.2‰ in darkness to -15.8‰ in the light. In Pinus monticola, the δC of CO efflux increased from -27.7‰ in darkness to -10.2‰ in the light. Observed increases in δC were generally in good agreement with predictions from the theoretical model. Second, we found that δC of dark-respired CO decreased following 2-3 h of illumination (P<0.01 for Populus tremuloides, P<0.001 for Pinus monticola). These decreases suggest that refixed photosynthate rapidly mixes into the respiratory substrate pool. Third, a field experiment demonstrated that bark photosynthesis influenced whole-tissue δC. Long-term light exclusion caused a localized increase in the δC of whole bark and current-year wood in branches of P. monticola (P<0.001 and P<0.0001, respectively). Thus bark photosynthesis was shown to discriminate against C and create a pool of photosynthate isotopically lighter than the dark respiratory pool in all three experiments. Failure to account for discrimination during bark photosynthesis could interfere with interpretation of the δC in woody tissues or in woody-tissue respiration.