Thiolutin was found to inhibit the utilization of glucose and other growth substrates in Escherichia coli. The inhibition was detected by a sharp drop of the respiration rate after addition of the antibiotic. The actual function affected was allocated to the cytoplasmic membrane of the bacterial cells by the following evidence: --spheroplasts were affected like intact cells, --individual reactions of either the electron transport chain or the glycolytic pathway were not inhibited, --glucose consumption in the culture stopped and the cells accumulated guanosine tetraphosphate as under starvation conditions, --activation of the cell's apo-glucose dehydrogenase restored respiration via bypassing the glucose phosphotransferase system. It was concluded that the transport of certain substrates across the membrane was inhibited.