Up-to-date, considerably less is known about the early stage in protein crystallization. By using AFM a stepwise process from the solution state to the regular aggregation in the early stage of Duck Cu, Zn superoxide dismutase (dSOD) crystallization has been observed. The process could be divided into four steps. At the beginning dSOD is in monodispersion as dimers. As supersaturation increases the irregular aggregation gives rise to linear polymers with different sizes and branches. When the system is nearing the maximum supersaturation the partial regular oligomerization happens to produce the oligomers with limited size, basically the nonamers of dSOD. In about 20 days after setup of the experiment regular assemblies with a double-helix-like pattern emerge from solution. Then the single crystals grow in about 45 days. The X-ray crystallographic analysis shows that the unique packing unit in the crystal is a double-helix formed by two strands of dSOD nonamers with 9(2) screw symmetry, which is well corresponding to the observation in the AFM images. The observations provide the experimental information for a better understanding of the process from solution to nucleation in protein crystallization. (C) 2002 Elsevier Science B.V. All rights reserved.