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Heterotrophic Nicotiana tabacum (L. CV. Wisconsin 38) plants are enriched in 13C relative to the carbon sources in their growth medium. We examined whether carboxylation via phosphoenolpyruvate carboxylase contributes to the enrichment. Achlorophyllous plants were produced using an inhibitor of carotenoid synthesis and were grown on sucrose with known delta(13)C values. Groups of plants were exposed to air with different delta(13)C values as well as to CO(2)-free air. The delta(13)C values of heterotrophic plants were greater than the sucrose source in all treatments and this enrichment increased as 13CO(2)/12CO(2) ratios increased in the source air. Rubisco activity was ruled out as a cause for the enrichment observed as 13CO(2)/12CO(2) ratios increased because the delta(13)C values of heterotrophic plants were similar when exposed to high 13CO(2) while grown in the light or dark. Neither was enrichment due to the adsorption of 13CO(2) in the high 13CO(2) treatment because dead plants did not exhibit this effect when subjected to the same atmospheric treatments. Carboxylation by PEP carboxylase is a likely mechanism causing the 13C-enriched values of living white tissues relative to their organic carbon sources. These results experimentally support suggestions that the anaplerotic activity of PEP is responsible for the 13C-enrichment commonly observed where heterotrophic inputs to growth are large such as in very young leaves.