Abstract: The Saccharomyces cerevisiae Snf1 kinase plays a critical role in recalibrating cellular metabolism in response to glucose depletion. Hundreds of genes show changes in expression levels when the SNF1 gene is deleted. However, cells can adapt to the absence of a specific gene when grown in long-term culture. Here we apply a chemical genetic method to rapidly and selectively inactivate a modified Snf1 kinase using a pyrazolopyrimidine inhibitor. By allowing cells to adjust to a change in carbon source prior to inhibition of the Snf1 kinase activity, we identified a set of genes whose expression increased when Snf1 was inhibited. Prominent in this set are genes that are activated by Gcn4, a transcriptional activator of amino acid biosynthetic genes. Deletion of Snf1 increased Gcn4 protein levels without affecting its mRNA levels. The increased Gcn4 protein levels required the Gcn2 kinase and Gcn20, regulators of GCN4 translation. These data indicate that Snf1 functions upstream of Gcn20 to regulate control of GCN4 translation in S. cerevisiae.