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Tree stem respiration (RS) is a substantial component of the forest carbon balance. The mass balance approach uses stem CO2 efflux and internal xylem fluxes to sum up RS, while the oxygen‐based method assumes O2 influx as a proxy of RS. So far, both approaches have yielded inconsistent results regarding the fate of respired CO2 in tree stems, a major challenge for quantifying forest carbon dynamics. We collected a data set of CO2 efflux, O2 influx, xylem CO2 concentration, sap flow, sap pH, stem temperature, nonstructural carbohydrates concentration and potential phosphoenolpyruvate carboxylase (PEPC) capacity on mature beech trees to identify the sources of differences between approaches. The ratio of CO2 efflux to O2 influx was consistently below unity (0.7) along a 3‐m vertical gradient, but internal fluxes did not bridge the gap between influx and efflux, nor did we find evidence for changes in respiratory substrate use. PEPC capacity was comparable with that previously reported in green current‐year twigs. Although we could not reconcile differences between approaches, results shed light on the uncertain fate of CO2 respired by parenchyma cells across the sapwood. Unexpected high values of PEPC capacity highlight its potential relevance as a mechanism of local CO2 removal, which merits further research. Summary Statement: Our field experiment in mature beech trees, measuring CO2 and O2 fluxes simultaneously, showed that 30% of the respired CO2 is retained in the stem. However, CO2 internal fluxes could not explain the difference between CO2 efflux and O2 influx. The internal carbon recycling mechanism mediated by PEPC is active in mature trees and can be considered as a missing C sink. [ABSTRACT FROM AUTHOR]