Many proteins are translocated through the SecY channel in bacteria and archaea and through the related Sec61 channel in eukaryotes(1). The channel has an hourglass shape with a narrow constriction approximately halfway across the membrane, formed by a pore ring of amino acids(2). While the cytoplasmic cavity of the channel is empty, the extracellular cavity is filled with a short helix called the plug(2), which moves out of the way during protein translocation(3,4). The mechanism by which the channel transports large polypeptides and yet prevents the passage of small molecules, such as ions or metabolites, has been controversial(2,5-8). Here, we have addressed this issue in intact Escherichia coli cells by testing the permeation of small molecules through wild-type and mutant SecY channels, which are either in the resting state or contain a defined translocating polypeptide chain. We show that in the resting state, the channel is sealed by both the pore ring and the plug domain. During translocation, the pore ring forms a 'gasket-like' seal around the polypeptide chain, preventing the permeation of small molecules. The structural conservation of the channel in all organisms indicates that this may be a universal mechanism by which the membrane barrier is maintained during protein translocation.