The value of an electron microscope equipped with a field emission gun (FEG) was first revealed in materials science applications. More recently, the FEG has played a crucial role in breaking the 10Angstrom barrier in single-particle reconstructions of frozen hydrated biological molecules. The standard high-resolution performance tests for electron microscopes are made close to focus, at several hundreds of Angstrom underfocus at a magnification of 500 000 x or more. While this is appropriate for materials science specimens, it is not suitable for observing frozen hydrated biological specimens with which the optimum underfocus is of the order of 1 micron or so and the magnification is limited by radiation damage to roughly 30 000 to 60 000 x. Thus, in order to access the performance of a cryo-electron microscope for high-resolution 3D electron microscopy of biological molecules, additional tests are necessary. We present here resolution tests of a 200-kV FEG using frozen hydrated virus suspensions. The extent and amplitude of the contrast transfer function are used as a test of the performance. We propose that small spherical viruses close to 300 Angstrom in diameter, such as the picornaviruses or phages, make good specimens for testing the performance of an electron microscope in cryo-mode. (C) 2002 Elsevier Science (USA). All rights reserved.