The largest terrestrial-to-atmosphere carbon flux is respired CO2. However, the partitioning of soil and plant sources, understanding of contributory mechanisms, and their response to climate change are uncertain. A plant removal experiment was established within a peatland located in the UK uplands to quantify respiration derived from recently-fixed plant carbon and that derived from decomposition of soil organic matter, using natural abundance 13C and bomb-14C as tracers. Soil and plant respiration sources were found respectively to contribute ~ 36% and between 41 and 54% of the total ecosystem CO2 flux. Respired CO2 produced in the clipped (‘soil’) plots had a mean age of ~ 15 years since fixation from the atmosphere, whereas the 14C content of ecosystem CO2 was statistically indistinguishable from the contemporary atmosphere. Results of carbon mass balance modelling showed that, in addition to respiration from bulk soil and plant respired CO2, a third, much older source of CO2 existed. This source, which we suggest is CO2 derived from the catotelm constituted between ~ 10 and 23% of total ecosystem respiration and had a mean radiocarbon age of between several hundred and ~ 2000 years before present (BP). These findings show that plant-mediated transport of CO2 produced in the catotelm may form a considerable component of peatland ecosystem respiration. The implication of this discovery is that current assumptions in terrestrial carbon models need to be re-evaluated to consider the climate sensitivity of this third source of peatland CO2.