Detailed information about unfolded states is required to understand how proteins fold. Knowledge about folding intermediates formed subsequently is essential to get a grip on pathological aggregation phenomena. During folding of apoflavodoxin, which adopts the widely prevalent alpha-beta parallel topology, most molecules fold via an off-pathway folding intermediate with helical properties. To better understand why this species is formed, guanidine hydrochloride-unfolded apoflavodoxin is characterized at the residue level using heteronuclear NMR spectroscopy. In 6.0 M denaturant, the protein behaves as a random coil. In contrast; at 3.4 M denaturant, secondary shifts and H-1-N-15 relaxation rates report four transiently ordered regions in unfolded apoflavodoxin. These regions have restricted flexibility on the (sub)nanosecond time scale. Secondary shifts show that three of these regions form a-helices, which are populated about 10% of the time, as confirmed by far-UV CID data. One region of unfolded apoflavodoxin adopts non-native structure. Of the a-helices observed, two are present in native apoflavodoxin as well. A substantial part of the third helix becomes beta-strand while forming native protein. Chemical shift changes due to amino acid residue replacement show that the latter a-helix has hydrophobic interactions with all other ordered regions in unfolded apoflavodoxin. Remarkably, these ordered segments dock non-natively, which causes strong competition with on-pathway folding. Thus, rather than directing productive folding, conformational preorganization in the unfolded state of an alpha-beta parallel-type protein promotes off-pathway species formation.