Coupling C-13 natural abundance and C-14 pulse labelling enabled us to investigate the dependence of C-13 fractionation on assimilate partitioning between shoots, roots, exudates, and CO2 respired by maize roots. The amount of recently assimilated C in these four pools was controlled by three levels of nutrient supply: full nutrient supply (NS), 10 times diluted nutrient supply (DNS), and deionised water (DW). After pulse labelling of maize shoots in a (CO2)-C-14 atmosphere, C-14 was traced to determine the amounts of recently assimilated C in the four pools and the delta C-13 values of the four pools were measured. Increasing amounts of recently assimilated C in the roots (from 8% to 10% of recovered C-14 in NS and DNS treatments) led to a 0.3 parts per thousand C-13 enrichment from NS to DNS treatments. A further increase of C allocation in the roots (from 10% to 13% of recovered 14C in DNS and DW treatments) resulted in an additional enrichment of the roots from DNS to DW treatments by 0.3 parts per thousand. These findings support the hypothesis that 13C enrichment in a pool increases with an increasing amount of C transferred into that pool. delta C-13 of CO2 evolved by root respiration was similar to that of the roots in DNS and DW treatments. However, if the amount of recently assimilated C in root respiration was reduced (NS treatment), the respired CO2 became 0.7 parts per thousand C-13 depleted compared to roots. Increasing amounts of recently assimilated C in the CO2 from NS via DNS to DW treatments resulted in a 1.6 parts per thousand delta C-13 increase of root respired CO2 from NS to DW treatments. Thus, for both pools, i.e. roots and root respiration, increasing amounts of recently assimilated C in the pool led to a delta C-13 increase. In DW and DNS plants there was no C-13 fractionation between roots and exudates. However, high nutrient supply decreased the amount of recently assimilated C in exudates compared to the other two treatments and led to a 5.3 parts per thousand C-13 enrichment in exudates compared to roots. We conclude that C-13 discrimination between plant pools and within processes such as exudation and root respiration is not constant but strongly depends on the amount of C in the respective pool and on partitioning of recently assimilated C between plant pools.