Electron cryotomography is the highest-resolution structural technique currently available that can be applied to unique objects such as flexible large protein complexes, irregular viruses, organelles and small cells. Specimens are preserved in a near-native, 'frozen-hydrated' state by vitrification. The thickness of the vitreous ice must be optimized for each specimen, and gold fiducials are typically added to facilitate image alignment. Here, we describe in detail our protocols for electron cryotomography sample preparation including (i) introduction of fiducial markers into the sample and (ii) sample vitrification. Because we almost exclusively use an automated, climate-controlled plunge-freezing device (the FEI Vitrobot) to vitrify our samples, we discuss its operation and parameters in detail. A session in which eight grids are prepared takes 1.5–2 h.