Abstract: Nutrient repletion leads to substantial restructuring of the transcriptome in Saccharomyces cerevisiae. Expression levels of approximately one third of all S. cerevisiae genes are altered by at least 2-fold when a nutrient-depleted culture is transferred to fresh medium. Several nutrient sensing pathways are known to play a role in this process, but the relative contribution that each pathway makes to the total response has not been determined. To better understand this, we used a chemical-genetic approach to block the PKA (protein kinase A), TOR (target of rapamycin), and glucose transport pathways, alone and in combination. Of the three pathways, we found that loss of PKA produced the largest effect on the transcriptional response; however, many genes required both PKA and TOR for proper nutrient regulation. Those genes that did not require PKA or TOR for nutrient regulation were dependent on glucose transport for either nutrient induction or repression. Therefore, loss of these three pathways is sufficient to prevent virtually the entire transcriptional response to fresh medium. In the absence of fresh medium, activation of the cAMP/PKA pathway does not induce cellular growth; nevertheless, PKA activation induced a substantial fraction of the PKA-dependent genes. In contrast, the absence of fresh medium strongly limited gene repression by PKA. These results account for the signals needed to generate the transcriptional responses to glucose, including induction of growth genes required for protein synthesis, repression of stress genes, as well as the classical glucose repression and hexose transporter responses.