This study investigates the effect of overexpression of key glycolytic enzymes exhibiting either native or alternative allosteric regulation on glucose bioconversion by resting Escherichia coli cells previously engineered for ethanol production. Homologous and heterologous pyruvate kinases (Pyk) and phosphofructokinases (Pfk) were individually and simultaneously overexpressed. Overexpression of the E. coli Pfk led to a shift from ethanol to lactate formation (threefold above the control level) while overexpression of Pyks accelerated lactate formation two-fold with less reduction in ethanol formation. Further increase in lactate formation (five-fold above the control level) resulted from overexpression of Pfk from Lactobacillus bulgaricus which, unlike the E. coli Pfk, is not allosterically regulated by either phosphoenolpyruvate or ADP. These effects on the carbon flux distribution were accompanied by significant changes in the intracellular concentrations of several glycolytic intermediates. Increased Pfk levels led primarily to reduced levels of hexose phosphates. Increased Pyk activity resulted in more complex changes which were different for overexpressed native Pyk and for overexpressed Bacillus stearothermophilus Pyk, which differs from E. coli Pyk in lacking activation by fructose 1,6-diphosphate, but is allosterically activated by AMP and ribose 5-phosphate. Simultaneous overexpression of native Pfk and Pyk caused a Pfk-overexpressionlike phenotype with lower levels of hexose phosphates and further increased lactate formation (nine-fold above the control level). The flux data demonstrate that overexpression of even single enzymes early in a central pathway can increase the fluxes to a particular metabolic product, although it may not affect the glucose uptake rate.