ABSTRACT Flux balance analysis and phenotypic data were used to provide clues to the relationships between the activities of gene products and the phenotypes resulting from the deletion of genes involved in respiratory function in Saccharomyces cerevisiae . The effect of partial or complete respiratory deficiency on the ethanol production and growth characteristics of hap4 Δ /hap4 Δ, mig1 Δ /mig1 Δ, qdr3 Δ /qdr3 Δ, pdr3 Δ /pdr3 Δ, qcr7 Δ /qcr7 Δ, cyt1 Δ /cyt1 Δ, and rip1 Δ /rip1 Δ mutants grown in microaerated chemostats was investigated. The study provided additional evidence for the importance of the selection of a physiologically relevant objective function, and it may improve quantitative predictions of exchange fluxes, as well as qualitative estimations of changes in intracellular fluxes. Ethanol production was successfully predicted by flux balance analysis in the case of the qdr3 Δ /qdr3 Δ mutant, with maximization of ethanol production as the objective function, suggesting an additional role for Qdr3p in respiration. The absence of similar changes in estimated intracellular fluxes in the qcr7 Δ /qcr7 Δ mutant compared to the rip1 Δ /rip1 Δ and cyt1 Δ /cyt1 Δ mutants indicated that the effect of the deletion of this subunit of complex III was somehow compensated for. Analysis of predicted flux distributions indicated self-organization of intracellular fluxes to avoid NAD + /NADH imbalance in rip1 Δ /rip1 Δ and cyt1 Δ /cyt1 Δ mutants, but not the qcr7 Δ /qcr7 Δ mutant. The flux through the glycerol efflux channel, Fps1p, was estimated to be zero in all strains under the investigated conditions. This indicates that previous strategies for improving ethanol production, such as the overexpression of the glutamate synthase gene GLT1 in a GDH1 deletion background or deletion of the glycerol efflux channel gene FPS1 and overexpression of GLT1 , are unnecessary in a respiration-deficient background.