Two equilibrium intermediates have previously been observed in the urea denaturation of the α subunit of tryptophan synthase (αTS) from Escherichia coli, an eight-stranded β/α barrel protein. In the current study, a series of amino-terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105–214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth β-strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of βαβ supersecondary structural elements. One-dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four βα units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein. These results suggest that the residual structure in αTS requires the participation of hydrophobic residues in multiple β-strands that span the entire sequence.