Gas–liquid mass transfer properties of shaken 96‐well microtiter plates were characterized using a recently described method. The maximum oxygen transfer capacity (OTR_max), the specific mass transfer area (a), and the mass transfer coefficient (k_L) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system. The shape (round and square cross‐sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters. To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter. The investigations revealed that the OTR_max is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round‐shaped wells. The mass transfer coefficient (k_L) in round‐shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface‐aerated bioreactors. The OTR_max in round‐shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material. Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed. This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area. This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks. In square‐shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTR_max, a, and k_L. An OTR_max of up to 0.15 mol/L/h can be reached in square‐shaped wells. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 178–186, 2003.