Vitreoscilla hemoglobin (VHb) has been expressed in Saccharomyces cerevisiae, and its influence on yeast aerobic metabolism has been investigated. New expression vectors were constructed to express VHb constitutively under the control of the ADH-1 promoter. The presence of VHb was shown by Western blot analysis. VHb has been shown to localize predominantly in the cytoplasm. Batch fermentation results indicated that the wild-type strain expressing VHb exhibited a shift in the carbon flux toward ethanol production, with no significant alteration in the specific growth rate. This effect was not observed if cells were grown under respiration inhibition, indicating that the metabolic effect of VHb is likely linked to respiration. Expression of VHb in the adh degrees strain MC65-2A, which produces ethanol only via a respiration-coupled pathway, revealed that ethanol production was decreased and cells reached a higher final cell density in a culture of the VHb-expressing strain. Growth enhancement due to expression of VHb was observed only during the final stage of culture growth when the acetaldehyde produced during the first growth phase was used as a substrate. This metabolic effect of intracellular VHb was seen more clearly in an acetaldehyde fed-batch fermentation in which VHb-expressing cells grew to at least 3-fold higher final cell density. These results suggest that the action of VHb is likely linked to electron transfer.