Lolium perenne, a main component species in managed grassland, is well adapted to defoliation, fertilization, and regrowth cycles; and hence, to changes in the assimilatory carbon source-sink ratio. In the Swiss Free Air CO2 Enrichment experiment the source-sink ratio is (i) increased by elevated partial pressure of CO2 (pCO2), (ii) decreased by enhanced carbon use under high N fertilization, and (iii) gradually increased during regrowth after defoliation. Since sucrose synthesis plays a central role in leaf carbohydrate metabolism in this fructan-accumulating species, we investigated how sucrose-phosphate synthase (SPS) responds to the differing assimilatory carbon fluxes and source-sink ratios in the field. Assimilatory carbon flux, as estimated by leaf gas exchange, strongly depended on pCO2. Surprisingly, the SPS content per leaf area did not increase with pCO2, but increased with N fertilization. During later regrowth, when a dense canopy had formed, the SPS content decreased; in particular, SPS was decreased at high N under elevated pCO2. Further, the higher assimilatory carbon flux through SPS at elevated pCO2 was accompanied by a higher activation state of SPS. The SPS content correlated very strongly with the ratio of free sucrose to free amino acid in leaves, which represents the carbon source-sink ratio. Hence, SPS content in L. perenne appears to be regulated by the current, strongly nitrogen-dependent, source-sink relation.