We have isolated cold-sensitive fermentation mutants (Csf mutants) of a commercial baker's yeast that have practically no fermentation capacity at 5°C and return to their normal capacity at 25 to 40°C. CSF1 was cloned by functional complementation of the Csf phenotype. CSF1 contain an open reading frame of 8,874 nucleotides, encoding a protein of 2,958 amino acids. The nucleotide sequence was identical to that of the YLR087C gene in the Saccharomyces genome database, but there was no information about the function of the predicted CSF1 (YLR087C) protein. Gene disruption shows that CSF1 is required for growth and fermentation only at low temperatures. Permeabilized cells of the disruptant showed nearly the same ethanol production rate as those of the parent strain, even at 10°C. The disruptant cells had the same glucose uptake rates as the parental cells at 30°C, but three- to fivefold-lower rates than the parental cells at 10°C. These findings suggest that CSF1 associates with a new nutrient transport system which exists on the plasma membrane and is required only at low temperature.