Using a genomic library constructed from Saccharomyces cerevisiae, we have identified a gene GFA1 that confers resistance to methylmercury toxicity. GFA1 encodes L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) and catalyzes synthesis of glucosamine-6-phosphate. Transformed yeast cells expressing GFA1 demonstrated resistance to methylmercury but no resistance to p-chloromercuribenzoate, a GFAT inhibitor. The cytotoxicity of methylmercury was inhibited by loading excess glucosamine 6-phosphate into yeast. Considering that GFAT is an essential cellular enzyme, our findings suggest that GFAT is the major target molecule of methylmercury in yeasts. This report is the first to identify the target molecule of methylmercury toxicity in eukaryotic cells.