During aerobic growth on glucose, Escherichia coli excretes acetate, a mechanism called "overflow metabolism." At high concentrations, the secreted acetate inhibits growth. Several mechanisms have been proposed for explaining this phenomenon, but a thorough analysis is hampered by the diversity of experimental conditions and strains used in these studies. Here, we describe the construction of a set of isogenic strains that remove different parts of the metabolic network involved in acetate metabolism. Analysis of these strains reveals that (i) high concentrations of acetate in the medium inhibit growth without significantly perturbing central metabolism ; (ii) growth inhibition persists even when acetate assimilation is completely blocked; and (iii) regulatory interactions mediated by acetyl-phosphate play a small but significant role in growth inhibition by acetate. The major contribution to growth inhibition by acetate may originate in systemic effects like the uncoupling effect of organic acids or the perturbation of the anion composition of the cell, as previously proposed. Our data suggest, however, that under the conditions considered here, the uncoupling effect plays only a limited role. IMPORTANCE High concentrations of organic acids such as acetate inhibit growth of Escherichia coli and other bacteria. This phenomenon is of interest for understanding bacterial physiology but is also of practical relevance. Growth inhibition by organic acids underlies food preservation and causes problems during high-density fermentation in biotechnology. What causes this phenomenon? Classical explanations invoke the uncoupling effect of acetate and the establishment of an anion imbalance. Here, we propose and investigate an alternative hypothesis: the perturbation of acetate metabolism due to the inflow of excess acetate. We find that this perturbation accounts for 20% of the growth-inhibitory effect through a modification of the acetyl phosphate concentration. Moreover, we argue that our observations are not expected based on uncoupling alone. KEYWORDS Escherichia coli, acetate, acetate metabolism, growth inhibition, metabolic flux analysis, overflow metabolism G rowth rate is probably the most important physiological parameter characterizing bacteria. The growth rate of a bacterial culture depends on the composition of the growth medium and the genotype of the particular strain. Under the most commonly used controlled growth condition, minimal medium supplemented with glucose as the sole carbon source, the model bacterium Escherichia coli secretes acetate, a by-product of glycolysis, during fast aerobic growth. This "overflow metabolism" is a function of growth rate. Experiments that vary the rate of glucose utilization by E. coli cells growing aerobically show a linear increase of growth rate, with the rate of glucose utilization up to around 0.6 h Ϫ1 (1). Beyond this growth rate, respiration becomes limiting at 15 mmol of O 2 per g of dry weight (gDW) and per h. Since glucose can no longer be fully oxidized to CO 2 , the extra redox potential is eliminated by secreting metabolites such as acetate