Abstract: Arsenic is ubiquitously present in nature and various mechanisms have evolved enabling cells to evade toxicity and acquire tolerance. Herein, we explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent arsenic (arsenite) by quantitative transcriptome, proteome and sulfur metabolite profiling. Arsenite exposure affected transcription of genes encoding functions related to protein biosynthesis, arsenic detoxification, oxidative stress defense, redox maintenance and proteolytic activity. Importantly, we observed that nearly all components of the sulfate assimilation and glutathione biosynthesis pathways were induced at both gene and protein levels. Kinetic metabolic profiling evidenced a significant increase in the pools of sulfur metabolites as well as elevated cellular glutathione levels. Moreover, the flux in the sulfur assimilation pathway as well as the glutathione synthesis rate strongly increased with a concomitant reduction of sulfur incorporation into proteins. By combining comparative genomics and molecular analyses, we pin-pointed transcription factors that mediate the core of the transcriptional response to arsenite. Taken together, our data reveals that arsenite-exposed cells channel a large part of assimilated sulfur into glutathione biosynthesis and we provide evidence that the transcriptional regulators Yap1p and Met4p control this response in concert. Key words: DNA microarray, proteome analysis, metabolite profiling, glutathione, arsenite.