Sucrose (Suc)-phosphate synthase (SPS) catalyses one of the rate-limiting steps in the synthesis of Suc in plants. The Arabidopsis genome contains four annotated SPS genes which can be grouped into three different families (SPSA1, SPSA2, SPSB, and SPSC). However, the functional significance of this multiplicity of SPS genes is as yet only poorly understood. All four SPS isoforms show enzymatic activity when expressed in yeast although there is variation in sensitivity towards allosteric effectors. Promoter-reporter gene analyses and quantitative real-time reverse transcription-PCR studies indicate that no two SPS genes have the same expression pattern and that AtSPSA1 and AtSPSC represent the major isoforms expressed in leaves. An spsa1 knock-out mutant showed a 44% decrease in leaf SPS activity and a slight increase in leaf starch content at the end of the light period as well as at the end of the dark period. The spsc null mutant displayed reduced Suc contents towards the end of the photoperiod and a concomitant 25% reduction in SPS activity. In contrast, an spsa1/spsc double mutant was strongly impaired in growth and accumulated high levels of starch. This increase in starch was probably not due to an increased partitioning of carbon into starch, but was rather caused by an impaired starch mobilization during the night. Suc export from excised petioles harvested from spsa1/spsc double mutant plants was significantly reduced under illumination as well as during the dark period. It is concluded that loss of the two major SPS isoforms in leaves limits Suc synthesis without grossly changing carbon partitioning in favour of starch during the light period but limits starch degradation during the dark period.