Twenty-seven deletion mutants of Saccharomyces cerevisiae were investigated using a novel method for high-content stoichiometric and kinetic metabolic profiling. Cultivation on glucose and galactose in 96-well microtiter plates with on-line optical sensing of dissolved oxygen was reproducible and identical to shake flask cultivation. At high shaking rate (1020 rpm) there was no oxygen limitation throughout all cultivations whereas at low shaking rate (540 rpm) oxygen uptake rate could be measured during declining dissolved oxygen concentration. This method allows the determination of specific growth rate, biomass yield on carbon substrate and oxygen and ethanol yield on carbon substrate. Maximum specific growth rates were in the same range for both sugars. On glucose the growth was predominantly fermentative with high yield of ethanol, low yield of biomass and low oxygen consumption rate. On galactose respiration was more active with correspondingly lower ethanol yields, higher biomass yields and higher rates of oxygen consumption. Some strains showed unexpectedly high or low growth rates and rates of ethanol production and respiration. Overall the four parameters determined for each mutant on two different carbon sources allowed statistically significant discrimination of all mutants studied. © 2006 Elsevier Ltd. All rights reserved.