Large oligomeric proteins are usually thought to fold and assemble hierarchically: Domains fold and coalesce to form the subunits, and folded subunits can then associate to form the multimeric structure. We have investigated the refolding pathway of the beta-sheet protein pea seed lectin using spectroscopic and hydrodynamic techniques. In vivo, it is proteolytically processed post-translationally, so that the single-domain subunits of the initial homodimer themselves become heterodimers of intertwined fragment polypeptide chains. Despite this complex topology, mature pea seed lectin reassembles with considerable efficiency at low total protein concentration (10 mug/mL) and low temperature (10 degrees C), albeit very slowly (t1/2 approximately 2 days). Contrary to expectations, the primary assembly product is not the intact beta-sheet domain, but the larger fragment chains first dimerize to form the native-like subunit interface. The smaller fragment chains then associate with this preformed dimer.