Efficient microbial conversion of biomass into renewable fuels and value-added chemicals remains an important goal in biotechnology. Xylose, which is the second most abundant sugar in nature and a major constituent of hemicellulose in lignocellulosic biomass and wastes, is an important substrate for these microbial processes. Escherichia coli expresses both a high-affinity, ATP-binding cassette xylose transporter (XylFGH), and a low-affinity proton symporter (XylE). The efficiency of xylose utilization in this organism is therefore suboptimal due to energetic requirements for xylose transport. E. coli has been engineered to reduce xylose to xylitol by expressing xylose reductase (XR). These studies showed that although xylose is negligibly metabolized by wild-type E. coli W3110 in the presence of glucose (classic diauxic growth), xylose transport and xylitol production do occur in the presence of glucose in wild-type E. coli expressing XR, and xylitol production is significantly improved in a mutant strain expressing cAMP-independent CRP (CRP*). These results indicated that either the native xylose transporters are not tightly controlled by CRP or additional xylose transport mechanisms exist. Overexpression of xylose transporters increases xylitol production in both wild-type and CRP* strains. E. coli strains carrying deletions in both native xylose transporters are still able to grow on xylose at rates approaching that of wild-type when using high concentrations of xylose (~100 mM), demonstrating that xylose is transported by at least one other, low-affinity system. Accurate modeling of xylose metabolism in E. coli in the presence of high concentrations of xylose thus requires a more thorough understanding of xylose uptake mechanisms and their energy requirements. Conversion of xylose to xylitol provides a measure of xylose transport without requiring xylose metabolism. This system allows studies of how various transporters influence xylose uptake under various conditions (e.g. in the presence of glucose) and not coupled to growth or expression of genes specific to xylose metabolism. Analysis of xylitol production in transporter mutants shows that xylose transport in the presence of glucose is not mediated by XylE or XylFGH (CRP control over these genes is tight), and secondary xylose transport is increased in the context of the crp* genotype. Up to 40% of xylose uptake was shown to occur via secondary transport, and this uptake is not affected by individual deletions in other transporters known to be promiscuous. Studies correlating anaerobic growth of engineered strains on xylose to energy yield indicate that secondary xylose transport is largely energy dependent (not diffusion). Radiolabeled xylose uptake studies are now helping to reveal kinetic properties of xylose transport in various mutant strains. These efforts toward a more complete understanding of xylose uptake in E. coli will be highlighted.