The range of value-added chemicals produced by Escherichia coli from simple sugars has been expanded to include xylitol. This was accomplished by screening the in vivo activity of a number of heterologous xylitol-producing enzymes. Xylose reductases from Candida boidinii (CbXR), Candida tenuis (CtXR), Pichia stipitis (PsXR), and Saccharmoyces cerivisiae (ScXR), and xylitol dehydrogenases from Gluconobacter oxydans (GoXDH) and Pichia stipitis (PsXDH) were all functional in E. coli to varying extents. Replacement of E. coli's native cyclic AMP receptor protein (CRP) with a cyclic AMP-independent mutant (CRP*) facilitated xylose uptake and xylitol production from mixtures of glucose and xylose, with glucose serving as the growth substrate and source of reducing equivalents. Of the enzymes tested, overexpression of NADPH-dependent CbXR produced the highest concentrations of xylitol in shake-flask cultures (∼275 mM in LB cultures, ∼180 mM using minimal medium). Expression of CbXR in strain PC09 (crp*, ΔxylB) in a 10-L controlled fermentation containing minimal medium resulted in production of ∼250 mM xylitol (38 g/L), with concomitant utilization of ∼150 mM glucose. The ratio of moles xylitol produced (from xylose) per mole glucose consumed was improved to >3.7:1 using metabolically active “resting” cells. © 2006 Wiley Periodicals, Inc.