Disorder in macromolecular crystals arises from several sources. Among the most important are thermal motion and the inherent mobility of the molecules. These cause statistical misalignment about mean lattice points and structural heterogeneity of the molecules. Defects in the crystal lattice, however, could also significantly affect the resolution and quality of diffraction data collected from some crystals. There is a considerable diversity in the sources of defects found in crystals, such as impurity incorporation and fluctuations of growth conditions, and the defects occur in a variety of structural forms. We have used in situ atomic force microscopy to visualize examples of several of the different classes of defects that occur in protein and virus crystals, ranging from unoccupied lattice sites (vacancies) to linear defects to stacking faults. In addition, we have calculated the defect density in several different macromolecular crystals and found that it varied in the range of 10(4)-10(6) cm(-2). Indeed, some preliminary evidence suggests that the ultimate resolution to which a crystal diffracts maybe a composite function of its inherent statistical disorder and its defect structure, density, and distribution. (C) 1996 Academic Press, Inc.