Pyruvate is a central metabolite that connects many metabolic pathways in living organisms. To meet the cellular pyruvate requirements, Escherichia coli most likely has three pyruvate uptake systems, BtsT, YhjX and CstA. Transport studies in intact cells proved that CstA is a high specific pyruvate transporter with moderate affinity, which is energized by a proton gradient. When the cells of a reporter strain were cultured in complex medium, cstA expression was bimodal, with the maximum being reached in the stationary phase. A DNA affinity capture assay combined with mass spectrometry and an in vivo reporter assay identified Fis as repressor in addition to the known cAMP-CRP as activator for cstA expression.