Differing claims regarding the stability of the recombinant ethanologen E. coli KO11 are addressed here in batch and chemostat culture. In repeat batch culture, the organism was stable on glucose, mannose, xylose and galactose for at least three serial transfers, even in the absence of a selective antibiotic. Chemostat cultures on glucose were remarkably stable, but on mannose, xylose and a xylose/glucose mixture, they progressively lost their hyperethanologenicity. On xylose, the loss was irreversible, indicating genetic instability. The loss of hyperethanologenicity was accompanied by the production of high concentrations of acetic acid and by increasing biomass yields, suggesting that the higher ATP yield associated with acetate production may foster the growth of acetate-producing revertant strains. Plate counts on high chloramphenicol-containing medium, whether directly, or following preliminary growth on non-selective medium, were not a reliable indicator of high ethanologenicity during chemostat culture. In batch culture, the organism appeared to retain its promise for ethanol production from lignocellulosics and concerns that antibiotics may need to be included in all media appear unfounded.