The role of fructose-2,6-bisphosphate (Fru-2,6-P-2) as a regulatory metabolite in photosynthetic carbohydrate metabolism was studied in transgenic Arabidopsis plants with reduced activity of Fru-6-phosphate,2-kinase/Fru-2,6-bisphosphatase. A positive correlation was observed between the Fru-6-Fhosyhate,2-kinase activity and the level of Fru-2,6-P-2 in the leaves. Thew partitioning of carbon was studied by (CO2)-C-14 labeling of photosynthetic products. Plant lines with Fru-2,G-P-2 levels down to 5% of the levels observed in wild-type (WT) plants had significantly altered partitioning of carbon between sucrose (Suc) versus starch. The ratio of C-14 incorporated into Suc and starch increased 2- to 3-fold in the plant's with low levels of Fru-2,6-P-2 compared with WT. Transgenic plant lines with intermediate levels of Fru-2,6-P-2 compared with WT had a Sue-to-starch labeling ratio similar to the WT. Levels of sugars, starch, and phosphorylated intermediates in leaves were followed during the diurnal cycle. Plants with low levels of Fru-2,6-P-2 in leaves had high levels of Feu-2,6-P-2 in leaves had high levels of Suc, glucose, and Fru and low levels of triose phosphates and glucose-1-P during the light period compared with WT. During the dark period these differences were eliminated. Our data provide direct evidence that Fru-2,6-P-2 affects photosynthetic carbon partitioning in Arabidopsis. Opposed to this, Fru-2,6-P-2 does not contribute significantly to regulation of metabolite levels in darkness.