Conditions are reported under which the reassembled intermediates of the heat-shock protein Hsp16.3 after being denatured in 8 M urea were detected by mainly using urea-gradient PAGE (with modifications) and urea-denaturing pore-gradient PAGE. Hsp16.3 is the small heat-shock protein from Mycobacterium tuberculosis, which exists as a specific nonamer and was proposed to form a trimer-of-trimers structure. The refolding and reassembling of this protein was achieved rapidly by dilution or dialysis, suggesting an effectively spontaneous recovery of quaternary structure. Data presented in this report demonstrate that the in vitro reassembling process of Hsp16.3 protein occurs through a spontaneous and effective stepwise mechanism. Modified urea-gradient PAGE may provide a general method for studying the reassembling processes of other oligomeric proteins.