UDP-glucose pyrophosphorylase (UGPase) produces UDP-glucose which is essential for sucrose and polysaccharide synthesis. Using Arabidopsis, we demonstrated that two UGPase genes (UGP1 and UGP2) are differentially expressed in a variety of organs, with UGP1 being pre-dominant. Co-expression analyses of UGP genes suggest that UGP1 is closely co-regulated with carbohydrate metabolism genes, late embryogenesis and seed loading, while UGP2 is co-regulated with stress response genes, fertilized flowers and photosynthetic genes. We have used Arabidopsis mutants for the UGP genes to characterize the role of both genes. The UGPase activity/protein was reduced by 70, 10 and 85% in ugp1, ugp2 and ugp1/ugp2 double mutant (DK) plants, respectively. A decrease in UGPase activity/protein was accompanied by an increase in expression of USP, a gene for UDP-sugar pyrophosphorylase, suggesting a compensatory mechanism. Generally, the mutants had no effects on soluble sugar/starch content (except in certain cases for DK plants), and there were no differences in cell wall composition/content between the wild type and the mutants. On the other hand, DK plants had greater hypocotyl and root lengths. When grown in the field, the mutants had as much as a 50% decrease in the number of seeds produced (consistent with a substantial decrease in field fitness), suggesting that they would be outcompeted in the field in a few generations. Overall, the data suggest that UGPase is not rate limiting for sucrose/starch and cell wall synthesis, but that it is essential in Arabidopsis.