The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permeation of nucleosides across the outer membrane under limiting substrate conditions. We purified Tsx from an E. coli strain that overproduces Tsx. The purified protein was still functional since it could neutralize the Tsx-specific bacteriophage T6 in vitro. When the purified Tsx was reconstituted into a lipid bilayer, there was a large increase of the membrane conductance, indicating pore-forming activity of Tsx in vitro. This increase could be strongly blocked with adenosine and to a much lesser extent with cytidine. Titration of the pore conductance with adenosine or cytidine suggested the presence of a binding site for nucleosides in the Tsx pore, with a Ks of 6 X 10(-4) and 2 X 10(-2) M for adenosine and cytidine, respectively. We propose that the Tsx protein functions in vivo as a pore that specifically facilitates the permeation of nucleosides across the outer membrane due to its binding site for nucleosides.