Soil respiration is the largest flux of carbon (C) from terrestrial ecosystems to the atmosphere. Here, we tested the hypothesis that photosynthesis affects the diurnal pattern of grassland soil-respired CO(2) and its C isotope composition (delta(13)C(SR)). A combined shading and pulse-labelling experiment was carried out in a mountain grassland. delta(13)C(SR) was monitored at a high time resolution with a tunable diode laser absorption spectrometer. In unlabelled plots a diurnal pattern of delta(13)C(SR) was observed, which was not explained by soil temperature, moisture or flux rates and contained a component that was also independent of assimilate supply. In labelled plots delta(13)C(SR) reflected a rapid transfer and respiratory use of freshly plant-assimilated C and a diurnal shift in the predominant respiratory C source from recent (i.e. at least 1 d old) to fresh (i.e. photoassimilates produced on the same day). We conclude that in grasslands the plant-derived substrates used for soil respiratory processes vary during the day, and that photosynthesis provides an important and immediate C source. These findings indicate a tight coupling in the plant-soil system and the importance of plant metabolism for soil CO(2) fluxes.