Tryptophan (Trp) fluorescence spectroscopy has been used to characterise interactions between the mechanosensitive channel of large conductance (MscL) of Mycobacterium tuberculosis (Tb) and its surrounding lipid bilayer. MscL is a homopentamer, each monomer containing two transmembrane α-helices, one lipid-exposed (TM2) and one lining the pore (TM1). Native MscL contains no Trp residues. Trp residues were incorporated into TbMscL at lipid-facing and pore-facing sites. Results from Trp-mutants of pore-lining residues suggest that water is able to penetrate into the channel pore, in the closed channel, upto the pore constriction formed by Thr-25. Trp residues located below Thr-25 and incorporated at helix-helix contact positions resulted in gain-of-function (GOF) mutants causing the channel to adopt an open conformation at zero tension. Reconstruction of Trp mutants of TbMscL into lipids of different chain lengths and different chain lengths and different head group structures resulted in no significant change in fluorescence emission maxima, suggesting that any changes in structure are small or result in no change in polarity for the Trp residue. Lipid-facing residues at the N-terminal end of TM1 are buried below the transmembrane surface of the protein. Fluorescence emission maxima for lipid-facing Trp residues in TM1 vary with position in the bilayer comparably to those for Trp residues in TM2, despite the fact that residues in TM2 are on the surface of the protein. Fluorescence emission spectra for most Trp residues on the periplasmic sides of TM1 and TM2 fit well to a model proposing a trough-like variation of dielectric constant across the membrane but the relationship between location and fluorescence emission maximum on the cytoplasmic side of the membrane is more complex. The fluorescence of Trp residues in TM1 is quenched efficiently by phospholipids with bromine-containing fatty acyl chains, showing that the lipid chains must be able to enter the Trp-containing cavities on the surface of MscL, resulting in efficient solvation of the surface.