Abstract: AMP-activated protein kinase, the "energy sensor of the cell," responds to low cellular energy stores by regulating enzymes and transcription factors that allow the cell to adapt to limiting nutritional conditions. Snf1, the yeast ortholog of AMP-activated protein kinase, has an essential role in respiratory metabolism in Saccharomyces cerevisiae that includes activating the transcription factor Adr1. How Snf1 regulates Adr1 activity is poorly understood. We used an analog-sensitive allele, SNF1(as)(I132G), that is inhibited by 2-naphthylmethyl pyrazolopyrimidine 1 to study the role of Snf1 in transcriptional regulation of glucose-repressible genes. When Snf1(as) was inhibited at the time of glucose depletion, there was a promoter-specific response with some Snf1-dependent genes being activated by low levels of inhibitor, whereas all Snf1-dependent genes were inhibited at high levels. Transcript accumulation was more sensitive to Snf1(as) inhibition than Adr1 or RNA polymerase (pol) II binding or acetylation of promoter nucleosomes. When Snf1(as) was inhibited after gene activation, Adr1 and RNA pol II remained at promoters, and RNA pol II remained in the ORF with associated nascent transcripts. However, cytoplasmic mRNAs were lost at a rapid rate compared with their decay following chemical or genetic inactivation of RNA pol II. In conclusion, Snf1 appears to affect multiple steps in gene regulation, including transcription factor binding, RNA polymerase II activity, and cytoplasmic mRNA stability.