The 303-residue cytolytic toxin ClyA forms a stable α-helical monomer. In the presence of detergents or membranes, however, the protein makes a large conformational transition to the protomer state, which is competent for assembly into a dodecameric cytolytic pore. In this study, we map the structure of the ClyA monomer during denaturant-induced unfolding with single-molecule Förster resonance energy transfer (FRET) spectroscopy. To this end, we probe intramolecular distances of six different segments of ClyA by placing donor and acceptor fluorophores at corresponding positions along the chain. We identify an intermediate state that contains the folded core consisting of three of the α-helices that make up the helical bundle present in the structure of both the monomer and the protomer, but with the C- and N-terminal helices unfolded, in accord with the secondary structure content estimated from circular dichroism (CD) spectroscopy. The existence of this intermediate is likely to be a consequence of the structural bistability underlying the biological function of ClyA: The terminal helices are part of the largest rearrangements during protomer formation, and the local differences in stability we detect may prime the protein for the required conformational transition.