AbstractUnder anaerobic conditions S. cerevisiae produces glycerol to regenerate NAD+ from the excess NADH produced in cell metabolism. We here report on the role of an uncharacterized protein, Yig1p (Ypl201cp), in anaerobic glycerol production. Yig1p was previously shown to interact in two‐hybrid tests with the GPP1 and GPP2 encoded glycerol 3‐phosphatase (Gpp), and we here demonstrate that strains overexpressing YIG1 show strongly decreased Gpp activity and content of the major phosphatase, Gpp1p. However, cells overexpressing YIG1 exhibited only slightly decreased GPP1 transcript levels, suggesting that Yig1p modulates expression on both transcriptional and post‐transcriptional levels. In agreement with such a role, a GFP‐tagged derivate of Yig1p was localized to both the cytosol and the nucleus. Deletion or overexpression of YIG1 did not, however, significantly affect growth yield or glycerol yield in anaerobic batch cultures, which is consistent with the previously proposed low flux control exerted at the Gpp level. Copyright © 2005 John Wiley & Sons, Ltd.