The expression pattern of the genes coding for vacuolar and extracellular invertase activity was analyzed in sugar beet (Beta vulgaris) and compared with the expression of sucrose synthase in this important sucrose-storing crop. Northern blot analysis revealed that sucrose synthase is the predominant sucrose-cleaving enzyme in tap roots, whereas vacuolar invertase was specifically expressed in petioles. Extracellular invertase transcripts showed low abundance in all the sugar beet organs and were not detected in northern blots. Relative RT-PCR analysis revealed differential expression of the two extracellular invertase genes: BVInv-CW1 was almost exclusively expressed in tap roots and BVInv-CW2 was widely expressed in all the organs analyzed. A remarkable result of this analysis was the high expression of vacuolar invertase (BVInv-V3) in petioles. Two factors had a clear influence on vacuolar invertase gene expression in petioles: light and the developmental stage, so that expression was higher in petioles from juvenile plants. BVInv-V3 transcripts showed circadian oscillation in petioles, with maximal accumulation during the light period. A similar pattern of diurnal oscillation was also observed for the vacuolar invertase activity, showing a delay with respect to the level of transcripts. The analysis of sugars in petioles revealed oscillation of the hexoses, with a remarkably higher content of glucose than fructose. In contrast, the level of sucrose in petioles was very low. This pattern of expression suggests an important role of petiole vacuolar invertase in plant development and photoassimilate partitioning.