Abstract The scanning transmission electron microscope (STEM) can be used for accurate and reproducible mass measurements. Here we analyse the major sources of systematic errors. Focus-dependent changes of the magnification can be corrected on-line by monitoring the objective-lens current. Post-specimen field effects are shown to be negligible for the Vacuum Generators STEM HB5 used. Operating conditions of the detector, a scintillator-photomultiplier combination, are critical and need to be calibrated for each experiment. The influence of sample purity, mass-loss kinetics and glutaraldehyde fixation on mass values is evaluated for several biological specimens, in particular for the widely used mass standard TMV. Possible errors arising from the use of mass standards to compensate for both instrumental and specimen-related uncertainties are considered.