Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, kLa, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. kLa values in one commercial reader were about 10 to 40 h−1. kLa values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate kLa values in a 96-well microtiter plate. The resulting values were about 130 h−1, which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h−1. Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 829–836, 2003.