Abstract Rhizosphere respiration can consume up to 30% of total net photosynthetic production, principally from root respiration and from rhizo-microbial respiration using plant-derived substrates. Rhizosphere respiration has rarely been studied in situ because of technical difficulties in partitioning respiration between roots and their rhizosphere associates. We report a procedure to simultaneously measure root respiration, rhizo-microbial respiration and soluble C concentration in the rhizospheres of intact plants. This procedure involves: (1) 14CO 2 pulse-labelling and tracing of photosynthetically-fixed C released from the root-soil column as 14CO 2 under three concentrations of [ 12C]glucose added to the soil; and (2) calculation of root respiration, rhizo-microbial respiration and soluble C concentrations in the rhizosphere. In an experiment with 3-week old wheat plants, we found that root respiration and rhizo-microbial respiration contributed, on average, 40.6 and 59.4% of total rhizosphere respiration, respectively, and that the soluble C concentration in the rhizosphere averaged 667 mg C 1 −1 of soil water. This soluble C concentration was about 20–100 times higher than the previous estimates used in some rhizosphere models.