Hexoses accumulate to high concentrations (∼ 200 mM) in storage parenchyma cells of tomato fruit. Hexoses are sourced from the fruit apoplasm as hydrolysis products of phloem-imported sucrose. Three hexose transporters (LeHT1, LeHT2, LeHT3), expressed in fruit storage parenchyma cells, may contribute to hexose uptake by these cells. An analysis of their full-length sequences demonstrated that all three transporters belong to the STP sub-family of monosaccharide transporters that localize to plasma membranes. Heterologous expression of LeHT1 (and previously LeHT2, Gear et al., 2000), but not LeHT3, rescued a hexose transport-impaired yeast mutant when raised on glucose or fructose as the sole carbon source. Biochemically, LeHT1, similarly to LeHT2, exhibited transport properties consistent with a high-affinity glucose/H(+) symporter. Significantly, LeHT1 and LeHT2 also functioned as low-affinity fructose/H(+) symporters with apparent K(m) values commensurate with those of fruit tissues. A substantial reduction (80-90%) in fruit expression levels of all LeHT genes by RNAi-mediated knockdown caused a 55% decrease in fruit hexose accumulation. In contrast, photoassimilate production by source leaves and phloem transport capacity to fruit were unaffected by transporter knockdown. Collectively, these findings demonstrate that LeHTs play key roles in driving accumulation of hexoses into storage parenchyma cells during tomato fruit development.