We describe a new approach for the simultaneous conversion of xylose and glucose sugar mixtures which potentially could be used for lignocellulosic biomass hydrolysate. In this study we used this approach to demonstrate the production of lactic acid. This process uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. In addition to knockouts in pflB encoding for pyruvate formate lyase, the xylose-selective (glucose deficient) strain E. coli ALS1073 has deletions of the glk, ptsG, and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1074 has a xylA deletion. By combining these two strains in a single process the xylose and glucose in a mixed sugar solution are simultaneously converted to lactate. Furthermore, the biomass concentrations of each strain can readily be adjusted in order to optimize the overall product formation. This approach to the utilization of mixed sugars eliminates the problem of diauxic growth, and provides great operational flexibility. Biotechnol. Bioeng. 2009; 102: 822–827. © 2008 Wiley Periodicals, Inc.