Escherichia coli produces lactate and acetate in significant amounts during both aerobic and anaerobic glycolysis. A model describing the mechanism of protein-mediated lactate transport has previously been proposed. A simple theoretical analysis here indicates that the proposed model would drain cellular energy resources by catalytically dissipating the proton-motive force. An experimental analysis of lactate and acetate transport employs nuclear magnetic resonance (NMR) spectroscopy to measure the relative concentrations of these end products on the two sides of the cytoplasmic membrane of anaerobically glycolyzing cells. Comparison of measured concentration ratios to those expected at equilibrium for various transport modes indicates that acetate is a classical uncoupling agent, permeating the membrane at comparable rates in the dissociated and undissociated forms. The lactate concentration ratio changes markedly after an initial period of sustained glycolysis. This change is most readily explained as resulting from a lactate transport system that responds to an indicator of glycolytic activity. The data further indicate that lactate permeates the membrane in both dissociated and undissociated forms. Both acids, then, are capable of catalytically dissipating the proton-motive force.