Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all carotenoids. IPP in Escherichia coli is synthesized through the nonmevalonate pathway, which has not been completely elucidated. The first reaction of IPP biosynthesis in E. coli is the formation of 1-deoxy-D-xylulose-5-phosphate (DXP), catalyzed by DXP synthase and encoded by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phos- phate, catalyzed by DXP reductoisomerase and encoded by dxr. To determine if one or more of the reactions in the nonmevalonate pathway controlled flux to IPP, dxs and dxr were placed on several expression vectors under the control of three different promoters and transformed into three E. coli strains (DH5alpha, XL1-Blue, and JM101) that had been engineered to produce lycopene. Lycopene production was improved significantly in strains transformed with the dxs expression vectors. When the dxs gene was expressed from the arabinose-inducible araBAD promoter (P(BAD)) on a medium-copy plasmid, lycopene production was twofold higher than when dxs was expressed from the IPTG-inducible trc and lac promoters (P(trc) and P(lac), respectively) on medium-copy and high-copy plasmids. Given the low final densities of cells expressing dxs from IPTG-inducible promoters, the low lycopene production was probably due to the metabolic burden of plasmid maintenance and an excessive drain of central metabolic intermediates. At arabinose concentrations between 0 and 1.33 mM, cells expressing both dxs and dxr from P(BAD) on a medium-copy plasmid produced 1.4-2.0 times more lycopene than cells expressing dxs only. However, at higher arabinose concentrations lycopene production in cells expressing both dxs and dxr was lower than in cells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-inducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XL1-Blue.