A 13C/12C mass spectrometer was interfaced with a open gas exchange system including four growth chambers to investigate CO2 exchange components of perennial ryegrass (Lolium perenne L.) stands. Chambers were fed with air containing CO2 with known δ13C (δCΟ2−2.6 or −46.8‰). The system did not fractionate C isotopes and no extraneous CO2 leaked into chambers. The on-line 13C discrimination (Δ) of ryegrass stands in light was independent of δCΟ2 when δCΟ2 was constant. The δ of CO2 exchanged by the stands in light (δNd) and darkness (δRn) differed by 0.7‰, suggesting some Δ in dark respiration at the stand-level. However, Δ decreased by ∼ 10‰ when δCΟ2 was switched from −46.8 to −2.5‰, and increased by ∼ 10‰ following a shift from −2.6 to −46.7‰ due to isotopic disequilibria between photosynthetic and respiratory fluxes. Isotopic imbalances were used to assess (non-photorespiratory) respiration in light and the replacement of the respiratory substrate pool(s) by new photosynthate. Respiration was partially inhibited by light, but increased during the light period and decreased in darkness, in association with temperature changes. The labelling kinetics of respiratory CO2 indicated the existence of two major respiratory substrate pools: a fast pool which was exchanged within hours, and a slow pool accounting for ∼ 60% of total respiration and having a mean residence time of 3.6 d.