The mechanosensitive channel of large conductance (MscL) is a non-selective ion channel which opens in response to changes in the transmembrane pressure profile, providing an important mechanism by which prokaryotes can respond to osmotic changes in their environment. The structure of this homo-pentameric ion channel has been well characterized in its closed state, but because it has not been crystallized in its open state, the details of its open structure remain somewhat unclear. The large distance involved prevents an accurate measurement of the size of the open pore using electron paramagnetic resonance. Ensemble fluorescence resonance energy transfer (FRET) measurements are not definitive because energy transfer between multiple donors and acceptors within the pentamer and between neighboring channels precludes direct interpretation of the data. We present a single-molecule FRET approach to measuring the diameter of the MscL open pore. A single cysteine mutation in the channel was labeled with an equal ratio of donor (AlexaFluor 488) to acceptor (Alexa 747) maleimides, but with a low overall ratio of fluorophores to monomers. The channel was reconstituted into small unilamellar vesicles at a protein to lipid ratio such that roughly one channel was inserted into each liposome. Measurements were made on the channels in the closed state, or while trapped in the open state by the introduction of lysophospholipids. Histograms were generated including only fluorescence traces in which both the donor and the acceptor each photobleach in a single-stepping pattern. These FRET histograms are used to determine changes in pore radius as the channel moves from closed to open states.