The in situ atomic force microscopy (AFM) investigation reveals the sources of disorder in crystals of an icosahedral plant virus, cucumber mosaic virus (CMV). It is here demonstrated that CMV virions that form a new 2D nuclei on (001) faces bind in arbitrary positions. This results in the formation of new growth layers with height differences in the range 0.5-2 nm. Coalescence of growth layers with height differences of up to 2 nm proceed without defect formation, while the merger of those with greater differences in height proceed with formation of domain boundaries. As a result, the (001) faces of hexagonal CMV crystals are formed by numerous misoriented domains with sizes in the range 0.5-50 mum(2). A variety of abnormal virions that have diverse sizes and that frequently cause defect formation were visualized in the CMV crystalline lattice. The combination of defects and local disorder presented here are likely the physical bases for mosaicity in crystals and may be largely responsible for their limited diffraction resolution. Studies of molecular mobility on the virus crystalline surface revealed the initial stages of two-dimensional nucleation as well as the mechanisms for virion incorporation into the step edges. The molecular structures of the step edges, which advance by the mechanism of one-dimensional (1D) nucleation, were recorded, and attachment rates of individual virions into growth steps were measured. From these data, the attachment frequencies were measured and the attachment probability for CMV crystallization was estimated to be approximately 10(-2).