Cytosolic fructose-1,6-bisphosphatase (cytFBPase) (E.C. 3.1.3.11) catalyzes the first irreversible reaction of the sucrose synthesis pathway, hydrolyzing fructose-1,6-bisphosphate (FBP) to yield fructose-6-phosphate (F6P) and inorganic phosphate (Pi). It is thought to be an essential enzyme for sucrose synthesis in the light. A 'Flaveria linearis' mutant ('FlcytFBPase ') previously characterized as having 10% of wildtype activity of cytFBPase, was shown to contain less than 1% cytFBPase activity. A F2 low cytFBPase population had an average cytFBPase activity that is 0% of wildtype. A western blot of 'FlcytFBPase' lacked the cytFBPase protein, and there was a deletion in the active site of 'FlcytFBPase' cDNA due to a point mutation in the genomic DNA. This is the only characterized null mutant known in plants that has a defect in an endogenous gene coding for an enzyme involved in daytime sucrose biosynthesis. Theoretically, null cytFBPase activity causes both high daytime starch accumulation and CO 2-insensitivity of photosynthesis. Although all lines with null cytFBPase activity exhibit CO2-insensitivity/reversed sensitivity at high CO2 concentrations, the null cytFBPase population has variable daytime starch accumulation phenotypes. Null cytFBPase activity causes reduced total capacity for end-product synthesis regardless of additional pathways affecting leaf sucrose-starch partitioning. If cytFBPase is essential for daytime sucrose synthesis, a null mutant should not grow in continuous light. Comparing growth of parental lines and progeny under continuous light versus a 16/8 h day/night regime indicates that the reduced dry matter accumulation phenotype of the null cytFBPase mutants is rescued under continuous light conditions. Collectively, the data indicate that alternative pathways for daytime sucrose synthesis exist.