The T-domain of diphtheria toxin senses a low pH to insert into a lipid membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Previous work has identified three transmembrane segments of T-domain in the open channel state, corresponding to TH5, TH8 and TH9 in the aqueous crystal structure; the amino-terminal region, TH1-TH4, was shown to be translocated across the membrane to the trans side. It was also shown that residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, so it was concluded that the TH6-TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of mutant T-domains with a single cysteine residue at positions in TH6-TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific methanethiosulfonate reagents on the channel conductance. For at least 12 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. We also compared the reaction rate of reagent added to the cis side vs. that to the trans side in order to estimate the residue's position along the channel axis. This analysis revealed abrupt changes in cis- vs. trans-side accessibility, suggesting that the TH6-TH7 segment forms a constriction that occupies a small portion of the total channel length. The location of this constriction relative to the TH8-TH9 segment was also determined.