Escherichia coli cells possess several mechanosensitive ion channels but only MscL, the channel with the highest conductance, which is activated at the highest membrane tension, has been cloned. We investigated the putative involvement of MscL in the effluxes caused by osmotic downshock. Osmotic shock caused the release of potassium glutamate, trehalose, and glycine betaine from wild type cells and cells lacking MscL. There was no difference between the two strains, but the extreme rapidity of the efflux process, as shown herein for glycine betaine, suggests that it is channel-mediated. Osmotic downshock also induces the release of some cytosolic proteins from EDTA-treated cells. We investigated the release of thioredoxin. This protein was totally released from wild type cells but was retained by MscL- cells. Release was restored by expression of the gene coding for MscL. Thus MscL is not necessary for the excretion of osmoprotectants, but it does open in vivo during shock and catalyzes the efflux of thioredoxin and possibly other small cytosolic proteins. It follows that the other mechanosensitive channels, which are known to be activated at lower tension, must also open during osmotic shock. Their opening and that of MscL could account for the rapid release of osmolytes.