In the present work, we report our recent progress in the development, optimization, and application of a technique for the three-dimensional (3-D) high-resolution characterization of crystalline microstructures. The technique is based on automated serial sectioning using a focused ion beam (FIB) and characterization of the sections by orientation microscopy based on electron backscatter diffraction (EBSD) in a combined FIB-scanning electron microscope (SEM). On our system, consisting of a Zeiss-Crossbeam FIB-SEM and an EDAX-TSL EBSD system, the technique currently reaches a spatial resolution of 100 · 100 · 100 nm 3 as a standard, but a resolution of 50 · 50 · 50 nm 3 seems to be a realistic optimum. The maximum observable volume is on the order of 50 · 50 · 50 lm 3 . The technique extends all the powerful features of two-dimensional (2-D) EBSD-based orientation microscopy into the third dimension of space. This allows new parameters of the microstructure to be obtained-for example, the full crystallographic characterization of all kinds of interfaces, including the morphology and the crystallographic indices of the interface planes. The technique is illustrated by four examples, including the characterization of pearlite colonies in a carbon steel, of twins in pseudonanocrystalline NiCo thin films, the description of deformation patterns formed under nanoindents in copper single crystals, and the characterization of fatigue cracks in an aluminum alloy. In view of these examples, we discuss the possibilities and limits of the technique. Furthermore, we give an extensive overview of parallel developments of 3-D orientation microscopy (with a focus on the EBSD-based techniques) in other groups.