Coupling growth of Lolium perenne L. in sterile solution culture with steady-state (13)CO(2) labelling allowed quantification of the contribution of C, assimilated either before or after a specific time point, both to plant compartments and root exudates. Plants were grown for 27 days in atmospheres containing CO(2) with delta(13)C signatures of either -13.5 or -36.1 per thousand. Air supplies to plants were then reciprocally switched to the opposing signature (day 0), plants were destructively harvested and root exudates collected over the next 8 days. Following the switch, C assimilated after day 0 and transported to the roots initially only appeared in root tips, later appearing in both tip and non-tip material. The delta(13)C signature of the root exudate changed exponentially with time. Assimilation pre- and post-day 0 contributed equally to exudate C at 4.5 days. Beyond day 8, assimilation pre-day 0 still contributed 41.7% of exudate C. Over all 8 days, a linear relationship existed between the delta(13)C signatures of root tips and exudate, suggesting that all newly assimilated C in the exudate was from root tips. Results imply pulse-labelling approaches to study root exudates are discriminative in the sources of exudates labelled and in the sites from which exudation occurs.