Vicinal diketones (VDK) cause butter-like off-flavors in beer and are formed by a non-enzymatic oxidative decarboxylation of alpha-aceto-alpha-hydroxybutyrate and alpha-acetolactate, which are intermediates in isoleucine and valine biosynthesis taking place in the mitochondria. On the assumption that part of alpha-acetolactate can be formed also in the cytosol due to a mislocalization of the responsible acetohydroxyacid synthase encoded by ILV2 and ILV6, functional expression in the cytosol of acetohydroxyacid reductoisomerase (Ilv5p) was explored. Using the cytosolic Ilv5p, I aimed to metabolize the cytosolically formed alpha-aetolactate, thereby lowering the total VDK production. Among mutant Ilv5p enzymes with varying degrees of N-terminal truncation, one with a 46-residue deletion (Ilv5p Delta 46) exhibited an unequivocal localization in the cytosol judged from microscopy of the Ilv5p Delta 46-green fluorescent protein fusion protein and the inability of Ilv5p Delta 46 to remedy the isoleucine/valine requirement of an ilv5 Delta strain. When introduced into an industrial lager brewing strain, a robust expression of Ilv5p Delta 46 was as effective as that of a wild-type Ilv5p in lowering the total VDK production in a 2-l scale fermentation trial. Unlike the case of the wild-type Ilv5p, an additional expression of Ilv5p Delta 46 did not alter the quality of the resultant beer in terms of contents of aromatic compounds and organic acids.