Escherichia coli strains devoid of one or both of the two pyruvate kinase isoenzymes (PKA and PKF), were grown on minimal media in batch fermentations. The strain lacking both PKs showed a 28% decrease on its specific growth rate when compared to the wild type. However, protein and CO2 yields did not change. Using radioactive 1-C-14 glucose and collecting the CO2 produced by the cultures, it was found that the mutant lacking both pyruvate kinases, metabolized glucose mainly through the pentose pathway (PP). The increased participation of the PP in glucose metabolism in this strain, was also reflected on the levels of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases.